Anti-Transglutaminase 2 Antibodies

ABSTRACT

The invention provides antibodies and antigen-binding fragments thereof that selectively bind to an epitope within the core region of transglutaminase type 2 (TG2). Novel epitopes within the TG2 core are provided. The invention provides human TG2 inhibitory antibodies and uses thereof, particularly in medicine, for example in the treatment and/or diagnosis of conditions including Celiac disease, scarring, fibrosis-related diseases, neurodegenerative/neurological diseases and cancer.

The present invention relates to inhibitors of TG2 and methods forproviding and using such inhibitors.

Transglutaminase type 2 (TG2; also known as tissue transglutaminase,tTg) is part of the wider 9 member transglutaminase family that includesFactor XIIIa, which is critical for blood clotting, as well asKeratinocyte Transglutaminase (TG1) and Epidermal Transglutaminase (TG3)that are involved in terminal differentiation of the keratinocyte. Inaddition, there are other TG family members such as TG types 4 to 7where no definitive role has been identified to date.

TG2 functions primarily as a protein cross-linking enzyme by catalysingthe formation of ε(γ-glutamyl) lysine iso-dipeptide bonds. Elevatedexpression of TG2 leads to aberrant protein cross-linking which has beenassociated with several pathologies including various types of tissuescarring, the formation of neurofibrillary tangles in several braindisorders and resistance to chemotherapy in some cancers. TG2 is alsoable to deamidate proteins. TG2 deamidates gliadin and the TG2/Gliadincomplex is the primary autoantigen in celiac disease. In addition, TG2has a GTP binding function and can act as a GTPase, although this hasnot been linked to a pathological role.

Elevated TG2 activity is primarily associated with abnormal woundhealing [1] leading to liver [2], pulmonary [3], heart [4] and kidneyfibrosis, [5] as well as atherosclerosis [6]. The process of scarringand fibrosis is linked to the increased synthesis and, most importantly,raised export of TG2 to the interstitial space. Once outside the cell,TG2 is able to crosslink extracellular matrix (ECM) proteins such asfibronectin and collagen [7] by the incorporation of an ε(γ-glutamyl)lysine di-peptide bond [8]. Studies have shown that this can acceleratethe deposition of available ECM components, while at the same timeconferring resistance to proteolytic clearance by the matrixmetalloproteinase (MMP) system [9, 10]. Taken together, this causes anaccumulation of ECM proteins and thus scar tissue [9]. Further, TG2 hasan emerging role in the activation of latent TGF-β1 in the scarringprocess [11] and has also been associated with interleukin-6 [12] andtumour necrosis factor-α activation pathways [13].

Inhibition of TG2 in vitro decreases extracellular matrix levels [14]while cells derived from TG2 knockout mice have lower levels of matureECM [9]. In vivo application of pan TG inhibitors in models of chronickidney disease reduce the development of glomerulosclerosis andtubulointerstitial fibrosis preserving renal function [15, 16]. Similarbenefits are seen in the TG2 knockout mouse subjected to unilateralureteric obstruction [17].

There are several neurodegenerative diseases characterised by thepresence of protein aggregates in the degenerative region of the brainthat TG2 has been implicated in forming. The best characterised is inHuntington's chorea. Huntington protein (htt) contains expandedpolyglutamine repeats in its N-terminal domain. Wild-type htt containsless than 35 consecutive glutamines while disease-related htt typicallyhas over 40 consecutive glutamines which makes it an excellent TG2substrate. Subsequently, insoluble aggregates are formed in the striatumand cortex of Huntington's disease patients. The frequency of aggregatescorrelates well with the severity of the disease.

Alzheimer's disease is typified by the presence of extracellular senileplaques composed of aggregated amyloid β-protein and intracellularneurofibrillary tangles consisting of a highly phosphorylated form ofthe protein tau. These plaques contain large amounts of ε(γ-glutamyl)lysine iso-dipeptide bonds.

Finally, a hallmark of Parkinson's disease is the presence ofalpha-synuclein aggregates called Lewy bodies in the cytoplasm ofaffected neurons which again contain ε(γ-glutamyl) lysine iso-dipeptidebonds. All of the aforementioned proteins are good substrates of TG2 invitro. Furthermore, the affected region of the brain contains higherlevels of TG2 protein than non-affected regions of the brain in the samepatients. The correlation between the TG2 substrate specificity fordisease-relevant aggregated proteins and increased TG2 expression levelssuggest a role for enzymatically active TG2 in each disease.

TG inhibitors have been shown to exert therapeutic effects in multiplebiological models of neurodegenerative diseases. In a cell culture modelof Parkinson's disease transfecting COS-7 cells with alpha-synuclein andTG2 simultaneously, covalent-synuclein aggregates, reminiscent of Lewybodies in Parkinson's disease, form and are dependent upon enzymaticallyactive TG2 since the C277S inactive TG2 mutant failed to induceaggregate formation. Treatment of these cotransfected cells withcystamine significantly reduced the quantity of alpha-synucleinaggregates as well as the percentage of cells containing the aggregates.There have been two other reports in which proteins with normal lengthand expanded polyglutamine repeat proteins, representative of expandedCAG diseases, such as Huntington's disease, have been transfected intocell lines and shown to form aggregates. Treatment of these cell lineswith the TG competitive inhibitor monodansylcadaverine led to a decreasein nuclear fragmentation, while treatment with cystamine lead to both adecrease in nuclear fragmentation, and a decrease in protein aggregateformation. An example of a pan TG inhibitor is1,3-Dimethyl-2-[(2-oxo-propyl)thio]imidazolium chloride which isavailable from Zedira GmbH and referred to in several publications asNTU283 or r283.

Cystamine has a beneficial therapeutic effect in vivo when dosed inmouse models of Huntington's disease. Huntington R6/2 mice dosed withcystamine showed improved motor function, less severe weight loss, andincreased survival compared to non-treated controls. Importantly, exvivo TG2 activity in brain homogenates was lower after dosing withcystamine at least 60 min after injection. In a different mouse model ofHuntington's disease, the YAC128 strain, cystamine was able to decreasethe level of striatal atrophy but unable to improve animal weight ormotor function, indicating a beneficial effect of cystamine at thecellular and tissue level but not in disease symptoms.

Probably the most convincing evidence that the beneficial therapeuticeffect of cystamine on Huntington mice is independent of TG2 inhibitionhas come from crossing the R6/2 Huntington mouse with the TG2 knockoutmouse to create a strain susceptible to neurodegeneration in the absenceof TG2. When the R6/2 TG2^(−/−) mice were treated with cystamine, theimproved motor function and increased lifespan were not statisticallydifferent from the improvement seen in R6/2 TG2^(+/+) mice treated withcystamine. Additionally, R6/1 and R6/2 TG2^(−/−) mice had increasedlevels of neuronal protein aggregates compared to R6/1 and R6/2TG2^(+/+) mice suggesting a mechanism of protein aggregation independentof TG2 transamidation activity in these models. However, it isnoteworthy that R6/2 TG2^(−/−) mice showed a delay in the onset of motordysfunction and improved survival compared to R6/2 TG2^(+/+) miceimplying a role for TG2 in the pathogenesis of neurodegeneration in theR6/2 model.

TG2 is also heavily implicated in celiac disease which affects 1 in 100people in Western Europe. Celiac sprue is a T cell-mediated inflammatorydisorder of the small intestine caused by a class of proteins calledprolamins found in wheat, barley, and rye. The high proline andglutamine content of these proteins makes them resistant to naturalgastric, pancreatic, and intestinal proteases, and peptidases duringdigestion. The resulting peptide fragments remain undigested well intothe small intestine and gain access to the intestinal lamina propriawhere, after modification by TG2, they can stimulate a T cell-mediatedimmune response leading to inflammation and destruction of intestinalarchitecture. Intestinal TG2 deamidates specific glutamine residues inthe prolamin peptides to glutamate residues. In HLA-DQ2/8 individualsthese modified peptides are presented to corresponding autoreactive Tcells by class II MHC molecules. Although prolamins have a highglutamine content (around 30-35%), only a few of these glutamineresidues are targeted by human TG2. An excellent correlation between TG2substrate specificity, DQ2 binding affinity, and T cell stimulatorypotential of TG2-treated prolamins strongly suggests that peptidedeamidation is mediated by TG2 and plays a significant role indetermining the severity of disease. Further, celiac patients generatean autoantibody response to TG2-gliadin complexes. These anti-TG2antibodies are found in both the small intestine, where they have beenshown to co-localize with extracellular TG2, and in the blood, wherethey are exploited as a diagnostic disease marker.

Despite the lack of animal models of celiac disease, ex vivo experimentsindicate that TG2 inhibition has the potential to benefit patients withceliac sprue. Culturing celiac patient small intestinal biopsies witheither TG2 treated (deamidated) or non-TG2 treated (non-deamidated)gluten digests both resulted in the generation of patient T-cell linesthat preferentially recognised deamidated gluten peptides rather thannon-deamidated gluten peptides. Also by blocking the activity ofendogenous TG2 in the celiac biopsies with cystamine more than half ofthe resultant T-cell lines had reduced proliferative responses comparedto non-cystamine-treated controls. Cell lines did not respond well tothe non-deamidated digests. These results imply that the glutenresponsive T-cell populations in celiac intestinal biopsies arenaturally biased towards recognizing deamidated gluten peptides asopposed to non-deamidated peptides, that endogenous TG2 activity inthese biopsies can result in gluten peptide deamidation in situ and thattreatment of celiac biopsies with TG2 inhibitors can reduce theproliferative response of gluten-reactive T cells.

Another study showed that the pan-TG inhibitor2-[(2-oxopropyl)thio]imidazolium inhibitor was able to prevent the insitu crosslinking of gluten peptides to endogenous proteins in thintissue sections taken from both celiac sprue patients and controls. Moreimportantly, the authors showed that incubation of intact celiac smallintestinal biopsies with 2-[(2-oxopropyl)thio]imidazolium preventedT-cell activation induced by the non-deamidated form of animmunodominant gluten peptide. In contrast, TG inhibition wasineffective at controlling T-cell activation when the biopsies wereincubated with the deamidated version of the same peptide. These resultssuggest that inhibition of endogenous TG2 in celiac patient biopsies canprevent gluten peptide deamidation and, therefore, reduce T-cellactivation.

Several observations support the hypothesis that TG2 plays a role in thedevelopment of certain types of cancer. Multiple studies have shown thatTG2 protein is up-regulated in cancerous tissue relative to healthytissue in cancers such as glioblastomas, malignant melanomas, andpancreatic ductal adenocarcinomas to name a few. A positive correlationbetween the chemotherapeutic resistance and metastatic potential ofcertain cancers with TG2 expression levels has been demonstrated, whilein certain cell types TG2 has been shown to exert anti-apoptotic effectson cells while siRNA down-regulation of TG2 protein expression levels ortreatment with TG2 inhibitors sensitizes these cells to apoptosis. Onthe other hand, there are also reports of the down-regulation of TG2expression in certain types of cancer [18]. Recently, it has been shownthat TG2 is a binding partner for GPR56, a protein down-regulated inhighly metastatic cancer cells, suggesting that TG2 can act as a tumorsuppressing protein through its interaction with GPR56 [18].

Current transglutaminase inhibitors fall into 3 main classes: 1)Competitive amine inhibitors (e.g. cystamine and spermine) that competewith natural amine substrates; 2) Reversible allosteric inhibitors suchas GTP and a newly discovered class of compound with a thieno[2,3-d]pyrimidin-4-one acylhydrazide backbone; and 3) Irreversible inhibitorsincluding 2-[(2-oxopropyl)thio]imidazolium derivatives (acetylate activesite cysteine), 3-halo-4,5-dihydroisoxazoles (form a stableiminothioether in the active site) and carbobenzyloxy-L-glutaminylglycine analogues with a variety of reactive moieties inserted.

Most have been used in the experimental systems above and shownbeneficial outcomes. However, none of these inhibitors show TG isoformspecificity as they all target the conserved catalytic triad within thetransglutaminase family catalytic core. Consequently, all potentiallyhave the disadvantage of co-inhibition of Factor XIIIa, TG1 and TG3,which effectively prevents their application in human disease due to theside effects that can be expected.

WO 2006/100679 describes a specific anti-TG2 antibody produced byrecombinant technology from samples collected from three adult celiacpatients with high anti-TG2 antibody titres.

Given the association of TG2 with numerous disease states and compellingdata from non specific inhibitors, there is a need for highly selectiveand high efficacy TG2 inhibitors with minimal off target effects.

The listing or discussion of an apparently prior-published document inthis specification should not necessarily be taken as an acknowledgementthat the document is part of the state of the art or is common generalknowledge.

In a first aspect the present invention provides an antibody or anantigen-binding fragment thereof that selectively binds to an epitopewithin the core region of transglutaminase type 2 (TG2).

In certain embodiments it is envisaged that the antibody orantigen-binding fragment thereof will selectively bind to an epitopewithin the core region of human TG2, rat TG2, and/or mouse TG2. In aparticularly preferred embodiment, the TG2 is human TG2.

The full amino acid sequences of human, rat and mouse TG2 can be foundunder the Genbank Accession numbers NM_004613, NM_019386.2 andNM_009373.3. The coding part of these sequences are as follows:

Human TG2 nucleotide sequence:atggccgaggagctggtcttagagaggtgtgatctggagctggagaccaatggccgagaccaccacacggccgacctgtgccgggagaagctggtggtgcgacggggccagcccttctggctgaccctgcactttgagggccgcaactacgaggccagtgtagacagtctcaccttcagtgtcgtgaccggcccagcccctagccaggaggccgggaccaaggcccgttttccactaagagatgctgtggaggagggtgactggacagccaccgtggtggaccagcaagactgcaccctctcgctgcagctcaccaccccggccaacgcccccatcggcctgtatcgcctcagcctggaggcctccactggctaccagggatccagctttgtgctgggccacttcattttgctcttcaacgcctggtgcccagcggatgctgtgtacctggactcggaagaggagcggcaggagtatgtcctcacccagcagggctttatctaccagggctcggccaagttcatcaagaacataccttggaattttgggcagtttgaagatgggatcctagacatctgcctgatccttctagatgtcaaccccaagttcctgaagaacgccggccgtgactgctcccgccgcagcagccccgtctacgtgggccgggtggtgagtggcatggtcaactgcaacgatgaccagggtgtgctgctgggacgctgggacaacaactacggggacggcgtcagccccatgtcctggatcggcagcgtggacatcctgcggcgctggaagaaccacggctgccagcgcgtcaagtatggccagtgctgggtcttcgccgccgtggcctgcacagtgctgaggtgcctgggcatccctacccgcgtcgtgaccaactacaactcggcccatgaccagaacagcaaccttctcatcgagtacttccgcaatgagtttggggagatccagggtgacaagagcgagatgatctggaacttccactgctgggtggagtcgtggatgaccaggccggacctgcagccggggtacgagggctggcaggccctggacccaacgccccaggagaagagcgaagggacgtactgctgtggcccagttccagttcgtgccatcaaggagggcgacctgagcaccaagtacgatgcgccctttgtctttgcggaggtcaatgccgacgtggtagactggatccagcaggacgatgggtctgtgcacaaatccatcaaccgttccctgatcgttgggctgaagatcagcactaagagcgtgggccgagacgagcgggaggatatcacccacacctacaaatacccagaggggtcctcagaggagagggaggccttcacaagggcgaaccacctgaacaaactggccgagaaggaggagacagggatggccatgcggatccgtgtgggccagagcatgaacatgggcagtgactttgacgtctttgcccacatcaccaacaacaccgctgaggagtacgtctgccgcctcctgctctgtgcccgcaccgtcagctacaatgggatcttggggcccgagtgtggcaccaagtacctgctcaacctcaacctggagcctttctctgagaagagcgttcctctttgcatcctctatgagaaataccgtgactgccttacggagtccaacctcatcaaggtgcgggccctcctcgtggagccagttatcaacagctacctgctggctgagagggacctctacctggagaatccagaaatcaagatccggatccttggggagcccaagcagaaacgcaagctggtggctgaggtgtccctgcagaacccgctccctgtggccctggaaggctgcaccttcactgtggagggggccggcctgactgaggagcagaagacggtggagatcccagaccccgtggaggcaggggaggaagttaaggtgagaatggacctgctgccgctccacatgggcctccacaagctggtggtgaacttcgagagcgacaagctgaaggctgtgaagggcttccggaatgtcatca ttggccccgcctaa HumanTG2 Amino Acid sequence:MAEELVLERCDLELETNGRDHHTADLCREKLVVRRGQPFWLTLHFEGRNYEASVDSLTFSVVTGPAPSQEAGTKARFPLRDAVEEGDWTATVVDQQDCTLSLQLTTPANAPIGLYRLSLEASTGYQGSSFVLGHFILLFNAWCPADAVYLDSEEERQEYVLTQQGFIYQGSAKFIKNIPWNFGQFEDGILDICLILLDVNPKFLKNAGRDCSRRSSPVYVGRVVSGMVNCNDDQGVLLGRWDNNYGDGVSPMSWIGSVDILRRWKNHGCQRVKYGQCWVFAAVACTVLRCLGIPTRVVTNYNSAHDQNSNLLIEYFRNEFGEIQGDKSEMIWNFHCWVESWMTRPDLQPGYEGWQALDPTPQEKSEGTYCCGPVPVRAIKEGDLSTKYDAPFVFAEVNADVVDWIQQDDGSVHKSINRSLIVGLKISTKSVGRDEREDITHTYKYPEGSSEEREAFTRANHLNKLAEKEETGMAMRIRVGQSMNMGSDFDVFAHITNNTAEEYVCRLLLCARTVSYNGILGPECGTKYLLNLNLEPFSEKSVPLCILYEKYRDCLTESNLIKVRALLVEPVINSYLLAERDLYLENPEIKIRILGEPKQKRKLVAEVSLQNPLPVALEGCTFTVEGAGLTEEQKTVEIPDPVEAGEEVKVRMDLLPLHMGLHKLVVNFESDKLKAVKGFRNVIIGPA* Rat TG2 Nucleotide sequence:Atggccgaggagctgaacctggagaggtgcgatttggagatacaggccaatggccgtgatcaccacacggccgacctgtgccaagagaaactggtgctgcggcgaggccagcgcttccggctgacactgtacttcgagggccgtggctatgaggccagcgtggacagacttacatttggtgccgtgaccggcccagatcccagtgaagaggcagggaccaaggcccgcttctcactgtctgacgatgtggaggagggatcctggtcagcctctgtgctggaccaacaggacaatgtcctctcgctgcagctctgcaccccagccaatgctcctgttggccagtaccgcctcagcctggagacttctactggctaccaaggctccagcttcatgctgggtcacttcatcctgctcttcaatgcctggtgcccagcggatgacgtgtacctagattcagaggcggagcgccgggaatacgtcctcacacagcagggcttcatctaccagggctctgtcaagttcatcaagagtgtgccttggaactttgggcagtttgaggatgggatcctggatgcctgcctgatgcttttggatgtgaaccccaagttcctgaaggaccgtagccgggactgctcacgacgcagcagtcccatctatgtgggccgcgtggtgagcggcatggtcaactgcaatgatgaccagggtgtgcttctgggtcgctgggacaacaattatggggacggtatcagtcccatggcctggattggcagcgtggacattctgcggcgctggaaggaacacggctgtcagcaagtgaagtatggccagtgctgggtgtttgcggcggtagcctgcacagtgctgcggtgccttggcatccctaccagagtggtgaccaactacaactccgcccacgaccagaacagcaacctgctcatcgagtacttccgaaacgagtacggggagctggagagcaacaagagcgagatgatctggaatttccactgctgggtggagtcctggatgaccaggccagacctacagccaggctatgaggggtggcaggccattgaccccacaccgcaggagaagagcgaaggaacatactgttgtggcccagtctcagtgcgggccatcaaggagggtgacctgagcaccaagtatgatgcgtccttcgtgtttgccgaggtcaacgctgatgtggtggactggatccggcagtcagatgggtctgtgctcaaatccatcaacaattccctggtcgtggggcagaagatcagcactaagagcgtgggccgtgatgaccgggaggacatcacctatacctacaagtacccagaggggtccccagaggagagggaagtcttcaccagagccaaccacctgaacaaactggcagagaaagaggagacaggggtggccatgcggatccgagtgggggatggtatgagcttgggcaatgactttgacgtgtttgcccacatcggcaacgacacctcggagagccgtgagtgccgcctcctgctctgtgcccgcactgtcagctacaacggcgtgctggggcccgagtgtggcactgaggacatcaacctgaccctggatccctactctgagaacagcatcccccttcgcatcctctacgagaagtacagcggttgcctgaccgagtcaaacctcatcaaggtgcggggtctcctcgtcgagccagccgctaacagctacctgctggctgagagagatctctacctggagaatcctgaaatcaagatccggatcctgggggagcccaagcagaaccgcaaactggtggctgaggtgtccctgaagaacccactttctgattccctgtatgactgtgtcttcactgtggagggggctggcctgaccaaggaacagaagtctgtggaggtctcagaccctgtgccagcaggagatgcggtcaaggtgcgggttgacctgttcccgactgatattggcctccacaagttggtggtgaacttccagtgtgacaagctgaagtcggtcaagggttaccggaatatcatcatcg gcccggcctaag Rat TG2Amino Acid sequence: MAEELNLERCDLEIQANGRDHHTADLCQEKLVLRRGQRFRLTLYFEGRGYEASVDRLTFGAVTGPDPSEEAGTKARFSLSDDVEEGSWSASVLDQQDNVLSLQLCTPANAPVGQYRLSLETSTGYQGSSFMLGHFILLFNAWCPADDVYLDSEAERREYVLTQQGFIYQGSVKFIKSVPWNFGQFEDGILDACLMLLDVNPKFLKDRSRDCSRRSSPIYVGRVVSGMVNCNDDQGVLLGRWDNNYGDGISPMAWIGSVDILRRWKEHGCQQVKYGQCWVFAAVACTVLRCLGIPTRVVTNYNSAHDQNSNLLIEYFRNEYGELESNKSEMIWNFHCWVESWMTRPDLQPGYEGWQAIDPTPQEKSEGTYCCGPVSVRAIKEGDLSTKYDASFVFAEVNADVVDWIRQSDGSVLKSINNSLVVGQKISTKSVGRDDREDITYTYKYPEGSPEEREVFTRANHLNKLAEKEETGVAMRIRVGDGMSLGNDFDVFAHIGNDTSESRECRLLLCARTVSYNGVLGPECGTEDINLTLDPYSENSIPLRILYEKYSGCLTESNLIKVRGLLVEPAANSYLLAERDLYLENPEIKIRILGEPKQNRKLVAEVSLKNPLSDSLYDCVFTVEGAGLTKEQKSVEVSDPVPAGDAVKVRVDLFPTDIGLHKLVVNFQCDKLKSVKGYRNIIIGPA*X Mouse TG2 Nucleotide sequence:atggcagaggagctgctcctggagaggtgtgatttggagattcaggccaatggccgtgaccaccacacggccgacctatgccaagagaaactggtgctgcgtcgtggtcagcgcttccggctgactctgtacttcgagggccgtggctacgaggccagcgtggacagcctcacgttcggtgctgtgaccggcccagatcccagtgaagaggcagggaccaaggcccgcttctcactgtctgacaatgtggaggagggatcttggtcagcctcagtgctggaccaacaggacaatgtcctctcgctacagctctgcaccccagccaatgctcctattggcctgtaccgtctcagcctagaggcttctactggctaccagggctccagctttgtgctgggccacttcatcctgctctacaatgcctggtgcccagccgatgatgtgtacctagactcagaggaggagcgacgggaatatgtccttacgcaacagggcttcatctaccaaggctctgtcaagttcatcaagagtgtgccttggaactttgggcagttcgaggatggaatcctggatacctgcctgatgctcttggatatgaaccccaagttcctgaagaaccgtagtcgggactgctcacgccgcagcagtcccatctatgtgggccgcgtggtgagcgccatggtcaactgcaatgatgaccagggtgtgcttctgggccgctgggacaacaactatggggatggtatcagtcccatggcctggattggcagtgtggacattctgcggcgctggaaggaacacggctgtcagcaagtgaagtacgggcagtgctgggtgtttgcagcggtggcctgcacagtgctgcggtgcctcggcatccctaccagagtggtgaccaactacaactccgcccacgaccagaacagcaacctgctcatcgagtacttccgaaatgagttcggggagctggagagcaacaagagcgagatgatctggaacttccactgctgggtggagtcctggatgaccaggccagacctacagccgggctatgaggggtggcaggccattgaccccacaccacaggagaagagcgaagggacatactgttgtggcccagtctcagtgcgagccatcaaggagggagacctgagtaccaagtatgatgcacccttcgtgtttgccgaggtcaacgctgatgtggtggactggatccggcaggaagatgggtctgtgctcaaatccatcaaccgttccttggtcgtggggcagaagatcagcactaagagtgtgggccgtgatgaccgggaggacatcacccatacctacaagtacccagaggggtcacccgaggagagggaagtcttcaccaaggccaaccacctgaacaaactggcagagaaagaggagacaggggtggccatgcgcatccgagtgggggacagtatgagcatgggcaacgacttcgacgtgtttgcccacatcggcaacgacacctcggagactcgagagtgtcgtctcctgctctgtgcccgcactgtcagctacaacggggtgctggggcccgagtgtggcactgaggacatcaacctgaccctggatccctactctgagaacagcatcccacttcgaatcctctacgagaagtacagcgggtgcctgacagagtcaaacctcatcaaggtgcggggccttctcatcgaaccagctgccaacagctacctgctggctgagagagatctctacctggagaatcccgaaatcaagatccgggtcctgggagaacccaagcaaaaccgcaaactggtggctgaggtgtccctgaagaacccactttccgatcccctgtatgactgcatcttcactgtggagggggctggcctgaccaaggagcagaagtctgtggaagtctcagacccggtgccagcgggcgatttggtcaaggcacgggtcgacctgttcccgactgatattggcctccacaagctggtggtgaacttccagtgtgacaagctgaagtcggtgaagggttaccggaatgttatcatcg gcccggcctaa Mouse TG2Amino Acid sequence: MAEELLLERCDLEIQANGRDHHTADLCQEKLVLRRGQRFRLTLYFEGRGYEASVDSLTFGAVTGPDPSEEAGTKARFSLSDNVEEGSWSASVLDQQDNVLSLQLCTPANAPIGLYRLSLEASTGYQGSSFVLGHFILLYNAWCPADDVYLDSEEERREYVLTQQGFIYQGSVKFIKSVPWNFGQFEDGILDTCLMLLDMNPKFLKNRSRDCSRRSSPIYVGRVVSAMVNCNDDQGVLLGRWDNNYGDGISPMAWIGSVDILRRWKEHGCQQVKYGQCWVFAAVACTVLRCLGIPTRVVTNYNSAHDQNSNLLIEYFRNEFGELESNKSEMIWNFHCWVESWMTRPDLQPGYEGWQAIDPTPQEKSEGTYCCGPVSVRAIKEGDLSTKYDAPFVFAEVNADVVDWIRQEDGSVLKSINRSLVVGQKISTKSVGRDDREDITHTYKYPEGSPEEREVFTKANHLNKLAEKEETGVAMRIRVGDSMSMGNDFDVFAHIGNDTSETRECRLLLCARTVSYNGVLGPECGTEDINLTLDPYSENSIPLRILYEKYSGCLTESNLIKVRGLLIEPAANSYLLAERDLYLENPEIKIRVLGEPKQNRKLVAEVSLKNPLSDPLYDCIFTVEGAGLTKEQKSVEVSDPVPAGDLVKARVDLFPTDIGLHKLVVNFQCDKLKSVKGYRNVIIGPA

The present inventors have raised antibodies to TG2 by immunising micewith a recombinant protein encompassing amino acids 143 to 473 of thehuman TG2 core. Hybridoma were screened for TG2 specificity andinhibition with any suitable candidates cloned. IgG was purified fromthese to calculate efficacy and the target epitope mapped by screeningof a human TG2 library by phage display.

The present approach to producing antibodies against TG2 by using arecombinant TG2 core protein has not been tried before and itsurprisingly led to the isolation and characterisation of antibodies toTG2 that were highly selective for TG2 and showed strong inhibitorycharacteristics. Prior attempts to raise antibodies to TG2 have led tothe isolation of relatively unselective antibodies that cross-react withother members of the transglutaminase family and thus would notrepresent promising antibodies for clinical use. The antibodies of thepresent invention on the other hand are promising candidates forclinical trials for diseases exacerbated by or mediated by TG2 activity.

It is surprising that the approach in the present application has led tothe production of much more effective antibodies than those previouslyproduced. There was no guarantee that it would have been possible toraise antibodies that are effective inhibitors of TG2 by immunising withthe core region. As indicated above, antibodies that are effectiveinhibitors may not be specific enough for TG2 to be used effectively inmedicine. It is surprising that antibodies to the divergent regions (inparticular, regions of the core that diverge slightly between differenttransglutaminase family members of TG2) are effective and selectiveinhibitors of TG2.

Without being bound by any theory we think that by raising antibodies toa smaller protein covering just the central core, we not only eliminatesome of the favoured immunological epitopes present on the full lengthprotein, but we also force core targeting. This appears to increase thevariety of antibodies available for selection and provides widercoverage of the core.

Immunising with just the TG2 core removed much of the tertiary structureof the enzyme (in particular the two carboxy terminal beta barreldomains). It is possible that some of the epitopes that perhaps may beless available or immunogenic within a native full length TG2 moleculemay be more attractive epitopes with the core in the format describedherein. The antibodies described herein recognised linear epitopes (i.e.bound to TG2 on a reducing SDS PAGE gel), whereas 80% of the antibodieswe previously isolated using full length TG2 as an immunogen wereconformation dependent. We were able to show that the recombinant coredomain retained enzyme activity, and so the isolation of inhibitoryantibodies was probably aided by the exposure of previously lessfavourable epitopes located in or near the active site. It isinteresting and surprising that inhibitory antibodies to human TG2 wereraised by immunising with the core given that the recombinant coreprotein may not have demonstrated the same folding characteristics asthe full length protein.

The TG catalytic core is highly conserved between members of the TGfamily and across species. This suggests that development of not onlyspecific small molecule inhibitors but also of antibody-based inhibitorsmay be technically challenging. Nevertheless, the present disclosureprovides antibodies that are highly selective. That this is possible mayreflect the fact that there are some regions within the TG2 catalyticdomain where there is some heterogeneity. The present antibodies maytherefore exploit these small differences. The surprising selectivity ofthe present antibodies may enable the development of therapeutics thatcan interfere efficiently with TG2 activity and thus provide potentiallyeffective therapies for conditions exacerbated by or caused by TG2activity where there is currently no satisfactory therapeutic option.

By way of comparison, the antibodies described in WO 2006/100679, whichwere produced by recombinant technology from samples from three adultceliac patients with high anti-TG2 antibody titres, proved to be of lowefficacy when tested by the present inventors (Example 2). Theantibodies of the present invention were far superior to those of WO2006/100679 in terms of selectivity for TG2 and in terms of inhibitoryactivity. For example, the present inventors generated a Fab fragment ofthe antibody of WO 2006/100679, which was applied at the sameconcentration in TG2 inhibition assays as the antibodies of the presentinvention. The Fab fragment amount in the tests was twice molar wisethat of the antibodies of the present invention, but they still failedto show any inhibition of TG2 activity. When the full length WO2006/100679 antibody was tested for inhibition in our standardputrescine incorporation assay no inhibition of TG2 activity was found.Thus, the methods of the present invention and the antibodies producedby those methods are superior to those previously described.

By “antibody” we include substantially intact antibody molecules, aswell as chimeric antibodies, humanised antibodies, human antibodies(wherein at least one amino acid is mutated relative to the naturallyoccurring human antibodies), single chain antibodies, bi-specificantibodies, antibody heavy chains, antibody light chains, homodimers andheterodimers of antibody heavy and/or light chains, and antigen bindingfragments and derivatives of the same. We also include variants, fusionsand derivatives of the antibodies and antigen-binding fragments thereofwithin the meaning of the terms “antibody” and “antigen-bindingfragments thereof”.

The term “antibody” also includes all classes of antibodies, includingIgG, IgA, IgM, IgD and IgE. Thus, the antibody may be an IgG molecule,such as an IgG1, IgG2, IgG3, or IgG4 molecule. Preferably, the antibodyof the invention is an IgG molecule, or an antigen-binding fragment, orvariant, fusion or derivative thereof. More preferably the antibody isan IgG2 molecule.

The antibodies, compositions, uses and methods of the inventionencompass variants, fusions and derivatives of the defined antibodiesand antigen-binding fragments thereof, as well as fusions of a saidvariants or derivatives, provided such variants, fusions and derivativeshave binding specificity for TG2.

As antibodies and antigen-binding fragments thereof comprise one or morepolypeptide component, variants, fusions and derivatives of the antibodyand antigen-binding fragment thereof as defined herein may be made usingthe methods of protein engineering and site-directed mutagenesis wellknown in the art using the recombinant polynucleotides (see example, seeMolecular Cloning: a Laboratory Manual, 3rd edition, Sambrook & Russell,2001, Cold Spring Harbor Laboratory Press, which is incorporated hereinby reference).

Thus, variants, fusions and derivatives of the antibody orantigen-binding fragment thereof as defined herein, may be made based onthe polypeptide component of the antibody or antigen-binding fragmentthereof.

By “fusion” we include said polypeptide fused to any other polypeptide.For example, the said polypeptide may be fused to a polypeptide such asglutathione-S-transferase (GST) or protein A in order to facilitatepurification of said polypeptide. Examples of such fusions are wellknown to those skilled in the art. Similarly, the said polypeptide maybe fused to an oligo-histidine tag such as His6 or to an epitoperecognised by an antibody such as the well-known Myc-tag epitope.Fusions to any variant or derivative of said polypeptide are alsoincluded in the scope of the invention. It will be appreciated thatfusions (or variants or derivatives thereof) which retain desirableproperties, such as have binding specificity for TG2, are preferred.

The fusion may comprise or consist of a further portion which confers adesirable feature on the said polypeptide; for example, the portion maybe useful in detecting or isolating the polypeptide, or promotingcellular uptake of the polypeptide. The portion may be, for example, abiotin moiety, a radioactive moiety, a fluorescent moiety, for example asmall fluorophore or a green fluorescent protein (GFP) fluorophore, aswell known to those skilled in the art. The moiety may be an immunogenictag, for example a Myc-tag, as known to those skilled in the art or maybe a lipophilic molecule or polypeptide domain that is capable ofpromoting cellular uptake of the polypeptide, as known to those skilledin the art.

By “variants” of said polypeptide we refer to a polypeptide wherein atone or more positions there have been amino acid insertions, deletions,and/or substitutions, either conservative or non-conservative, providedthat such changes result in a protein whose basic properties, forexample binding properties, thermostability, activity in a certainpH-range (pH-stability) have not significantly been changed.“Significantly” in this context means that one skilled in the art wouldsay that the properties of the variant may still be different but wouldnot be unobvious over the ones of the original protein. Thus, we includevariants of the polypeptide where such changes do not substantiallyalter the activity of the said polypeptide. In particular, we includevariants of the polypeptide where such changes do not substantiallyalter the binding specificity for TG2.

By “conservative substitutions” is intended combinations such as Gly,Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe,Tyr.

Such variants may be made using the methods of protein engineering andsite-directed mutagenesis.

The polypeptide variant may have an amino acid sequence which has atleast 75% identity with one or more of the amino acid sequences givenherein, for example at least 80%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identity with one ormore of the amino acid sequences specified herein.

The percent sequence identity between two polypeptides may be determinedusing suitable computer programs, for example the GAP program of theUniversity of Wisconsin Genetic Computing Group and it will beappreciated that percent identity is calculated in relation topolypeptides whose sequences have been aligned optimally.

The alignment may alternatively be carried out using the Clustal Wprogram (as described in Thompson et al., 1994, Nucl. Acid Res.22:4673-4680, which is incorporated herein by reference).

The parameters used may be as follows:

-   -   Fast pair-wise alignment parameters: K-tuple (word) size; 1,        window size; 5, gap penalty; 3, number of top diagonals; 5.        Scoring method: x percent.    -   Multiple alignment parameters: gap open penalty; 10, gap        extension penalty; 0.05.    -   Scoring matrix: BLOSUM.

Alternatively, the BESTFIT program may be used to determine localsequence alignments.

The antibody, antigen-binding fragment, variant, fusion or derivativeused in the methods or uses of the invention may comprise or consist ofone or more amino acids which have been modified or derivatised.

Chemical derivatives of one or more amino acids may be achieved byreaction with a functional side group. Such derivatised moleculesinclude, for example, those molecules in which free amino groups havebeen derivatised to form amine hydrochlorides, p-toluene sulphonylgroups, carboxybenzoxy groups, t-butyloxycarbonyl groups, chloroacetylgroups or formyl groups. Free carboxyl groups may be derivatised to formsalts, methyl and ethyl esters or other types of esters and hydrazides.Free hydroxyl groups may be derivatised to form O-acyl or O-alkylderivatives. Also included as chemical derivatives are those peptideswhich contain naturally occurring amino acid derivatives of the twentystandard amino acids. For example: 4-hydroxyproline may be substitutedfor proline; 5-hydroxylysine may be substituted for lysine;3-methylhistidine may be substituted for histidine; homoserine may besubstituted for serine and ornithine for lysine. Derivatives alsoinclude peptides containing one or more additions or deletions as longas the requisite activity is maintained. Other included modificationsare amidation, amino terminal acylation (e.g. acetylation orthioglycolic acid amidation), terminal carboxylamidation (e.g. withammonia or methylamine), and the like terminal modifications.

It will be further appreciated by persons skilled in the art thatpeptidomimetic compounds may also be useful. Thus, by ‘polypeptide’ weinclude peptidomimetic compounds which are capable of binding to anepitope within the TG2 core region. The term ‘peptidomimetic’ refers toa compound that mimics the conformation and desirable features of aparticular peptide as a therapeutic agent.

For example, the said polypeptide includes not only molecules in whichamino acid residues are joined by peptide (—CO—NH—) linkages but alsomolecules in which the peptide bond is reversed. Such retro-inversopeptidomimetics may be made using methods known in the art, for examplesuch as those described in Meziere et al. (1997) J. Immunol. 159,3230-3237, which is incorporated herein by reference. This approachinvolves making pseudo-peptides containing changes involving thebackbone, and not the orientation of side chains. Retro-inversepeptides, which contain NH—CO bonds instead of CO—NH peptide bonds, aremuch more resistant to proteolysis. Alternatively, the said polypeptidemay be a peptidomimetic compound wherein one or more of the amino acidresidues are linked by a -y(CH₂NH)— bond in place of the conventionalamide linkage.

In a further alternative, the peptide bond may be dispensed withaltogether provided that an appropriate linker moiety which retains thespacing between the carbon atoms of the amino acid residues is used; itmay be advantageous for the linker moiety to have substantially the samecharge distribution and substantially the same planarity as a peptidebond.

It will be appreciated that the said polypeptide may conveniently beblocked at its N- or C-terminus so as to help reduce susceptibility toexo-proteolytic digestion.

A variety of un-coded or modified amino acids such as D-amino acids andN-methyl amino acids have also been used to modify mammalian peptides.In addition, a presumed bioactive conformation may be stabilised by acovalent modification, such as cyclisation or by incorporation of lactamor other types of bridges, for example see Veber et al., 1978, Proc.Natl. Acad. Sci. USA 75:2636 and Thursell et al., 1983, Biochem.Biophys. Res. Comm. 111:166, which are incorporated herein by reference.

A common theme among many of the synthetic strategies has been theintroduction of some cyclic moiety into a peptide-based framework. Thecyclic moiety restricts the conformational space of the peptidestructure and this frequently results in an increased specificity of thepeptide for a particular biological receptor. An added advantage of thisstrategy is that the introduction of a cyclic moiety into a peptide mayalso result in the peptide having a diminished sensitivity to cellularpeptidases.

Thus, exemplary polypeptides useful in the methods and uses of theinvention comprise or consist of terminal cysteine amino acids. Such apolypeptide may exist in a heterodetic cyclic form by disulphide bondformation of the mercaptide groups in the terminal cysteine amino acidsor in a homodetic form by amide peptide bond formation between theterminal amino acids. As indicated above, cyclising small peptidesthrough disulphide or amide bonds between the N- and C-terminuscysteines may circumvent problems of specificity and half-life sometimeobserved with linear peptides, by decreasing proteolysis and alsoincreasing the rigidity of the structure, which may yield higherspecificity compounds. Polypeptides cyclised by disulphide bonds havefree amino and carboxy-termini which still may be susceptible toproteolytic degradation, while peptides cyclised by formation of anamide bond between the N-terminal amine and C-terminal carboxyl andhence no longer contain free amino or carboxy termini. Thus, thepeptides can be linked either by a C—N linkage or a disulphide linkage.

The present invention is not limited in any way by the method ofcyclisation of peptides, but encompasses peptides whose cyclic structuremay be achieved by any suitable method of synthesis. Thus, heterodeticlinkages may include, but are not limited to formation via disulphide,alkylene or sulphide bridges. Methods of synthesis of cyclic homodeticpeptides and cyclic heterodetic peptides, including disulphide, sulphideand alkylene bridges, are disclosed in U.S. Pat. No. 5,643,872, which isincorporated herein by reference. Other examples of cyclisation methodsare discussed and disclosed in U.S. Pat. No. 6,008,058, which isincorporated herein by reference.

A further approach to the synthesis of cyclic stabilised peptidomimeticcompounds is ring-closing metathesis (RCM). This method involves stepsof synthesising a peptide precursor and contacting it with an RCMcatalyst to yield a conformationally restricted peptide. Suitablepeptide precursors may contain two or more unsaturated C—C bonds.

The method may be carried out using solid-phase-peptide-synthesistechniques. In this embodiment, the precursor, which is anchored to asolid support, is contacted with a RCM catalyst and the product is thencleaved from the solid support to yield a conformationally restrictedpeptide.

Another approach, disclosed by D. H. Rich in Protease Inhibitors,Barrett and Selveson, eds., Elsevier (1986), which is incorporatedherein by reference, has been to design peptide mimics through theapplication of the transition state analogue concept in enzyme inhibitordesign. For example, it is known that the secondary alcohol of stalinemimics the tetrahedral transition state of the scissile amide bond ofthe pepsin substrate.

In summary, terminal modifications are useful, as is well known, toreduce susceptibility by proteinase digestion and therefore to prolongthe half-life of the peptides in solutions, particularly in biologicalfluids where proteases may be present. Polypeptide cyclisation is also auseful modification because of the stable structures formed bycyclisation and in view of the biological activities observed for cyclicpeptides.

Thus, in one embodiment the said polypeptide is cyclic. However, in analternative embodiment, the said polypeptide is linear.

By “selectively binds to an epitope within the core region of TG2” wemean an antibody or antigen-binding fragment thereof that is capable ofbinding to an epitope in the TG2 core region selectively. By “capable ofbinding selectively” we include such antibody-derived binding moietieswhich bind at least 10-fold more strongly to the TG2 core than to otherproteins; for example at least 50-fold more strongly, or at least100-fold more strongly. The binding moiety may be capable of bindingselectively to an epitope in the TG2 core under physiologicalconditions, e.g. in vivo.

Such binding specificity may be determined by methods well known in theart, such as enzyme-linked immunosorbent assay (ELISA),immunohistochemistry, immunoprecipitation, western blot and flowcytometry using transfected cells expressing TG2 or the TG2 core, or afragment thereof. Suitable methods for measuring relative bindingstrengths include immunoassays, for example where the binding moiety isan antibody (see Harlow & Lane, “Antibodies: A Laboratory Manual”, ColdSpring Harbor Laboratory Press, New York, which is incorporated hereinby reference). Alternatively, binding may be assessed using competitiveassays or using Biacore® analysis (Biacore International AB, Sweden).

It is preferred that the antibody or antigen-binding fragment of theinvention binds exclusively to TG2.

It will be appreciated by persons skilled in the art that the bindingspecificity of an antibody or antigen binding fragment thereof isconferred by the presence of complementarity determining regions (CDRs)within the variable regions of the constituent heavy and light chains.As discussed below, in a particularly preferred embodiment of theantibodies and antigen-binding fragments thereof defined herein, bindingspecificity for TG2 is conferred by the presence of one or more of theCDRs identified. For example, sequences that may comprise or consist ofthe CDR sequences of AB-1 VL and VH include KASQDINSYLT, RTNRLFD,LQYDDFPYT, SSAMS, TISVGGGKTYYPDSVKG, and LISLY. In a further example,sequences that may comprise or consist of the CDR sequences of BB-7 VLand VH include KASQDINSYLT, LTNRLMD, LQYVDFPYT, SSAMS,TISSGGRSTYYPDSVKG, and LISPY. Sequences that may comprise or consist ofthe CDR sequences of DC-1 VL and VH include KASQDINSYLT, LVNRLVD,LQYDDFPYT, THAMS, TISSGGRSTYYPDSVKG, and LISTY. It is preferred that theantibodies and antigen-binding fragments thereof defined herein compriseor consist of CDR sequences, or CDR and flanking sequences, as definedin Table 24A. It is most preferred that the antibodies andantigen-binding fragments thereof defined herein comprise or consist ofthe CDR sequences, or CDR and flanking sequences, of the exemplaryantibody AB-1, or BB-7, or DC-1 (as defined, for example, in Table 24A).

It is preferred that the antibody or antigen-binding fragment thereofretains the binding specificity for TG2 of the original antibody. By“retains the binding specificity” we mean that the antibody orantigen-binding fragment thereof is capable of competing for binding toTG2 with the exemplary antibodies of the invention, for example AB-1,AG-1, AG-9, AH-1, AH-3, BB-7, DC-1, EH-6, JE-12, IA-12, DF-4, DH-2, DD-6and/or DD-9 (see accompanying Examples). For example, the antibody orantigen-binding fragment thereof may bind to the same epitope on TG2 asan antibody comprising the following sequences: KASQDINSYLT, RTNRLFD,LQYDDFPYT, SSAMS, TISVGGGKTYYPDSVKG, and LISLY.

By “epitope” it is herein intended to mean a site of a molecule to whichan antibody binds, i.e. a molecular region of an antigen. An epitope maybe a linear epitope, which is determined by e.g. the amino acidsequence, i.e. the primary structure, or a three-dimensional epitope,defined by the secondary structure, e.g. folding of a peptide chain intobeta sheet or alpha helical, or by the tertiary structure, e.g. waywhich helices or sheets are folded or arranged to give athree-dimensional structure, of an antigen.

Methods for determining whether a test antibody is capable of competingfor binding with second antibody are well known in the art (such as, forexample sandwich-ELISA or reverse-sandwich-ELISA techniques) anddescribed, for example, in Antibodies: A Laboratory Manual, Harlow &Lane (1988, CSHL, NY, ISBN 0-87969-314-2), which is incorporated hereinby reference.

The antibody or antigen-binding fragment thereof, with bindingspecificity for an epitope in the TG2 core region may also retain one ormore of the same biological properties as the original antibody (such asthe exemplary antibodies provided in the Examples).

As explained above, TG2 is a calcium-dependent multi-functional proteinthat catalyzes the formation of Nε-(γ-glutamyl)lysine isopeptide bondsbetween lysine and glutamine residues. TG2 comprises an N terminal betasandwich domain that contains binding sites (e.g. fibronectin) andsequences required for enzyme export. This links to the catalytic coredomain. Central to the domain is a catalytic triad consisting of Cys277, Asp 358, and His 355, plus several putative calcium binding sites.This links to the third domain, beta barrel 1 where a GTP binding siteresides conveying the enzyme with GTPase activity. Beta barrel 1 alsocontains an integrin binding site used in cell adhesion. Beta Barrel 1along with the fourth TG2 domain, Beta barrel 2, are involved in theconformational change in TG2 required for its activation. In a highcalcium, low GTP environment Barrel 1 and 2 swing down from the closedand folded inactive form to convey TG2 with a linear structure openingup the catalytic core allowing activation (Pinkas et al (2007) PLoSBiol. Transglutaminase 2 undergoes a large conformational change uponactivation. 5(12): e327).

By “the core region of transglutaminase type 2 (TG2)” we include aregion of TG2 comprising the catalytic triad described above, excludingthe beta sandwich domain and beta barrels 1 and 2. Preferably the coreregion comprises or consists of amino acids 143 to 473 of human TG2, ora fragment thereof.

Amino acids 143 to 473 of human TG2 consist of the following sequence:

CPADAVYLDSEEERQEYVLTQQGFIYQGSAKFIKNIPWNFGQFEDGILDICLILLDVNPKFLKNAGRDCSRRSSPVYVGRVVSGMVNCNDDQGVLLGRWDNNYGDGVSPMSWIGSVDILRRWKNHGCQRVKYGQCWVFAAVACTVLRCLGIPTRVVTNYNSAHDQNSNLLIEYFRNEFGEIQGDKSEMIWNFHCWVESWMTRPDLQPGYEGWQALDPTPQEKSEGTYCCGPVPVRAIKEGDLSTKYDAPFVFAEVNADVVDWIQQDDGSVHKSINRSLIVGLKISTKSVGRDEREDITHTYKYPEGSSEEREAFTRANHLNKLAEKEETGM

Thus, in an embodiment of the invention, the core region may consist ofamino acids 143 to 473 of human TG2. In this embodiment, the epitope ofthe antibody of the invention could thus be any epitope within theregion defined by amino acids 143 to 473 of human TG2. Thus, the epitopemay be a fragment of this sequence or it could be made up of variousamino acid residues within this fragment that may not be adjacent oneanother in the primary amino acid structure but localise with oneanother in the secondary, tertiary or even quaternary structure of theprotein, as would be understood by a person of skill in the art.

In an embodiment of the invention the antibody or antigen-bindingfragment thereof may selectively bind in whole or in part to a regioncomprising amino acids 304 to 326 of human TG2. This region (amino acids304 to 326 of human TG2) is referred to as Group 1 in FIG. 5 andcomprises the amino acid sequence AHDQNSNLLIEYFRNEFGEIQGD.

In a further embodiment, the antibody or antigen-binding fragmentthereof may selectively bind in whole or in part to a region comprisingamino acids 351 to 365 of human TG2. This region (amino acids 351 to 365of human TG2) is referred to as Group 2 in FIG. 5 and comprises theamino acid sequence YEGWQALDPTPQEKS.

In a yet further embodiment of the invention, the antibody orantigen-binding fragment thereof may selectively bind in whole or inpart to a region comprising amino acids 450 to 467 of human TG2. Thisregion (amino acids 450 to 467 of human TG2) is referred to as Group 3in FIG. 5 and comprises the amino acid sequence SEEREAFTRANHLNKLAE.

In a preferred embodiment of any aspect of the invention, the antibodyor antigen-binding fragment thereof inhibits TG2 activity, for examplehuman TG2 activity.

In an embodiment of the invention the antibody or antigen-bindingfragment thereof may comprise one or more of the following amino acidsequences:

KASQDINSYLT; and/or RTNRLFD; and/or LQYDDFPYT; and/or SSAMS; and/orTISVGGGKTYYPDSVKG; and/or LISLY.

The antibody or antigen-binding fragment thereof of the invention maycomprise one or more of the following amino acid sequences:

TCKASQDINSYLTWF; and/or TLIYRTNRLFDGVP or TLIYRTNRLFDGVPXXFSGSGSGQDFF;and/or YCLQYDDFPYTFG; and/or FTLSSSAMSWVR or CXAXXFTLSSSAMSWVR; and/orWVATISVGGGKTYYPDSVKGRFTISR or WVATISVGGGKTYYPDSVKGRFTISRXNXXXXL; and/orYCAKLISLYWG, wherein X is any amino acid.

The sequences of the immediately preceding embodiments are considered tocomprise the complementarity determining regions of the light and heavyvariable regions of the exemplary antibody AB-1 (see Example 1) andcertain specified humanised variants of the AB-1 antibody. In a furtherembodiment, the antibody may comprise the amino acid sequenceKASQDINSYLTXXXXXXXXXXXXXXXRTNRLFDXXXXXXXXXXXXXXXFXXXXXXXXXXXXXXXXLQYDDFPYT; or KASQDINSYLTXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXF;or RTNRLFDXXXXXXXXXXXXXXF; or FXXXXXXXXXXXXXXXXLQYDDFPYT, wherein X isany amino acid.

In a further embodiment, the antibody or antigen-binding fragmentthereof may comprise the amino acid sequence TCKASQDINSYLTWF orTCKASQDINSYLTWY; and/or LLIYRTNRLFDGVP or SLIYRTNRLFDGVP orLLIYRTNRLFDGVPXXFSGSGSGQDFF or SLIYRTNRLFDGVPXXFSGSGSGQDFF; and/orYCLQYDDFPYTFG; and/or FTFSSSAMSWVR or CXAXXFTFSSSAMSWVR; and/orWVSTISVGGGKTYYPDSVKGRFTISR or WVSTISVGGGKTYYPDSVKGRFTISRXNXXXXL; and/orYCAKLISLYWG, wherein X is any amino acid.

Thus, in an embodiment, the antibody or antigen-binding fragment thereofmay have at least one light chain variable region comprising thefollowing sequences: KASQDINSYLT; and RTNRLFD; and LQYDDFPYT.

In a further embodiment, the antibody or antigen-binding fragmentthereof may have at least one heavy chain variable region comprising thefollowing sequences: SSAMS; and TISVGGGKTYYPDSVKG; and LISLY.

In an embodiment of the invention the antibody or antigen-bindingfragment thereof may have at least one light chain variable regioncomprising the amino acid sequenceDIQMTQTPSSMYASLGERVTITCKASQDINSYLTWFQQKPGKSPKTLIYRTNRLFDGVPSRFSGSGSGQDFFLTISSLEYEDMGIYYCLQYDDFPYTFGGGTKLEIK orDIKMTQSPSSMYASLGERVTITCKASQDINSYLTWFQQKPGKSPKTLIYRTNRLFDGVPSRFSGSGSGQDFFLTISSLEYEDMGIYYCLQYDDFPYTFGGGTKLEIK. These light chainvariable regions correspond with that found in exemplary antibody AB-1(FIGS. 7 and 18).

Alternatively, the antibody or antigen-binding fragment thereof may haveat least one light chain variable region comprising the amino acidsequence:

EIVLTQSPSSMYASLGERVTITCKASQDINSYLTWFQQKPGKSPKTLIYRTNRLFDGVPSRFSGSGSGQDFFLTISSLEYEDMGIYYCLQYDDFPYTFGG GTKLEIK (AB-1_VK) orDIQMTQSPSSMYASLGERVTITCKASQDINSYLTWFQQKPGKSPKTLIYRTNRLFDGVPSRFSGSGSGQDFFLTISSLEYEDMGIYYCLQYDDFPYTFGG GTKLEIK (AB-1_VK1).

In a particularly preferred embodiment the antibody or antigen-bindingfragment thereof may have at least one light chain variable regioncomprising the amino acid sequence:

EIVLTQSPSSLSASVGDRVTITCKASQDINSYLTWYQQKPGKAPKLLIYRTNRLFDGVPSRFSGSGSGTDFFFTISSLQPEDFGTYYCLQYDDFPYTFGG GTKLEIK (hAB-1_RKE);or DIQMTQSPSSLSASVGDRVTITCKASQDINSYLTWFQQKPGKAPKSLIYRTNRLFDGVPSRFSGSGSGTDFFLTISSLQPEDFATYYCLQYDDFPYTFGQ GTKVEIK (hAB-1_RKJ).

These sequences are humanised variants of the AB-1 light chain sequencesprovided above.

In an embodiment of the invention, the antibody or antigen-bindingfragment thereof may have at least one heavy chain variable regioncomprising the amino acid sequenceEVQLEESGGGLVKPGGSLKLSCAASGFTLSSSAMSWVRQTPDRRLEWVATISVGGGKTYYPDSVKGRFTISRDNAKNTLYLQMNSLRSEDTAMYYCAKLISLYWGQGTTLTVSS orEVQLVESGGGLVKPGGSLKLSCAASGFTLSSSAMSWVRQTPDRRLEWVATISVGGGKTYYPDSVKGRFTISRDNAKNTLYLQMNSLRSEDTAMYYCAKLISLYWGQGTTLTVSS. These heavychain variable regions correspond with that found in exemplary antibodyAB-1 (FIGS. 7 and 18).

Alternatively, the antibody or antigen-binding fragment thereof may haveat least one heavy chain variable region comprising the amino acidsequence:

EVQLQESGGGLVKPGGSLKLSCAASGFTLSSSAMSWVRQTPDRRLEWVATISVGGGKTYYPDSVKGRFTISRDNAKNTLYLQMNSLRSEDTAMYYCAKLI SLYWGQGTTLTVSS(AB-1_VH).

In a particularly preferred embodiment, the antibody or antigen-bindingfragment thereof may have at least one heavy chain variable regioncomprising the amino acid sequence:EVQLLESGGGLVQPGGSLRLSCAASGFTFSSSAMSWVRQAPGKGLEWVSTISVGGGKTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKLISLYWGQGTLVTVSS (hAB-1_RHA). Thissequence is a humanised variant of the AB-1 heavy chain sequenceprovided above.

Thus, it is envisaged that in an embodiment, the antibody orantigen-binding fragment thereof may have:

i) at least one light chain variable region comprising the amino acidsequence DIQMTQTPSSMYASLGERVTITCKASQDINSYLTWFQQKPGKSPKTLIYRTNRLFDGVPSRFSGSGSGQDFFLTISSLEYEDMGIYYCLQYDDFPYTFGG GTKLEIK orDIKMTQSPSSMYASLGERVTITCKASQDINSYLTWFQQKPGKSPKTLIYRTNRLFDGVPSRFSGSGSGQDFFLTISSLEYEDMGIYYCLQYDDFPYTFGG GTKLEIK, orEIVLTQSPSSMYASLGERVTITCKASQDINSYLTWFQQKPGKSPKTLIYRTNRLFDGVPSRFSGSGSGQDFFLTISSLEYEDMGIYYCLQYDDFPYTFGG GTKLEIK (AB-1_VK), orDIQMTQSPSSMYASLGERVTITCKASQDINSYLTWFQQKPGKSPKTLIYRTNRLFDGVPSRFSGSGSGQDFFLTISSLEYEDMGIYYCLQYDDFPYTFGG GTKLEIK (AB-1_VK1),or EIVLTQSPSSLSASVGDRVTITCKASQDINSYLTWYQQKPGKAPKLLIYRTNRLFDGVPSRFSGSGSGTDFFFTISSLQPEDFGTYYCLQYDDFPYTFGG GTKLEIK (hAB-1_RKE),or DIQMTQSPSSLSASVGDRVTITCKASQDINSYLTWFQQKPGKAPKSLIYRTNRLFDGVPSRFSGSGSGTDFFLTISSLQPEDFATYYCLQYDDFPYTFGQ GTKVEIK (hAB-1_RKJ);and ii) at least one heavy chain variable region comprising the aminoacid sequence EVQLEESGGGLVKPGGSLKLSCAASGFTLSSSAMSWVRQTPDRRLEWVATISVGGGKTYYPDSVKGRFTISRDNAKNTLYLQMNSLRSEDTAMYYCAKLI SLYWGQGTTLTVSS orEVQLVESGGGLVKPGGSLKLSCAASGFTLSSSAMSWVRQTPDRRLEWVATISVGGGKTYYPDSVKGRFTISRDNAKNTLYLQMNSLRSEDTAMYYCAKLI SLYWGQGTTLTVSS, orEVQLQESGGGLVKPGGSLKLSCAASGFTLSSSAMSWVRQTPDRRLEWVATISVGGGKTYYPDSVKGRFTISRDNAKNTLYLQMNSLRSEDTAMYYCAKLI SLYWGQGTTLTVSS(AB-1_VH), or EVQLLESGGGLVQPGGSLRLSCAASGFTFSSSAMSWVRQAPGKGLEWVSTISVGGGKTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKLI SLYWGQGTLVTVSS(hAB-1_RHA).

In an embodiment of any aspect of the present invention the antibody orantigen-binding fragment thereof may comprise one or more of thefollowing amino acid sequences:

KASQDINSYLT; and/or LTNRLMD; and/or LQYVDFPYT; and/or SSAMS; and/orTISSGGRSTYYPDSVKG; and/or LISPY.

The sequences of the immediately preceding embodiment are considered tocomprise the complementarity determining regions of the light and heavyvariable regions of the exemplary antibody BB-7 (see FIG. 19). In afurther embodiment, the antibody may comprise the amino acid sequenceKASQDINSYLTXXXXXXXXXXXXXXXLTNRLMDXXXXFXXXXXXXXXXX XXXXXXLQYVDFPYT; orKASQDINSYLTXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXF, orLTNRLMDXXXXXXXXXXXXXXF; or FXXXXXXXXXXXXXXXXXLQYVDFPYT, wherein X is anyamino acid.

Thus, in an embodiment of any aspect of the present invention, theantibody or antigen-binding fragment thereof may have at least one lightchain variable region comprising the following sequences: KASQDINSYLT;and LTNRLMD; and LQYVDFPYT.

In a further embodiment of any aspect of the present invention, theantibody or antigen-binding fragment thereof may have at least one heavychain variable region comprising the following sequences: SSAMS; andTISSGGRSTYYPDSVKG; and LISPY.

In an embodiment of any aspect of the present invention the antibody orantigen-binding fragment thereof may have at least one light chainvariable region comprising the amino acid sequenceAIKMTQSPSSMYASLGERVIITCKASQDINSYLTWFQQKPGKSPKTLIYLTNRLMDGVPSRFSGSGSGQEFLLTISGLEHEDMGIYYCLQYVDFPYTFGGGTKLEIK. This light chainvariable region corresponds with that found in exemplary antibody BB-7(FIG. 19).

In an embodiment of any aspect of the present invention, the antibody orantigen-binding fragment thereof may have at least one heavy chainvariable region comprising the amino acid sequenceAVQLVESGGGLVKPGGSLKLSCAASGIIFSSSAMSWVRQTPEKRLEWVATISSGGRSTYYPDSVKGRFTVSRDSAKNTLYLQMDSLRSEDTAIYYCAKLISPYWGQGTTLTVSS. This heavy chainvariable region corresponds with that found in exemplary antibody BB-7(FIG. 19).

Thus, it is envisaged that in an embodiment of any aspect of the presentinvention, the antibody or antigen-binding fragment thereof may have:

i) at least one light chain variable region comprising the amino acidsequence AIKMTQSPSSMYASLGERVIITCKASQDINSYLTWFQQKPGKSPKTLIYLTNRLMDGVPSRFSGSGSGQEFLLTISGLEHEDMGIYYCLQYVDFPYTFGG GTKLEIK; and ii) atleast one heavy chain variable region comprising the amino acid sequenceAVQLVESGGGLVKPGGSLKLSCAASGIIFSSSAMSWVRQTPEKRLEWVATISSGGRSTYYPDSVKGRFTVSRDSAKNTLYLQMDSLRSEDTAIYYCAKLI SPYWGQGTTLTVSS.

It is further envisaged that in an embodiment of any aspect of thepresent invention, the antibody or antigen-binding fragment thereof mayhave:

i) at least one light chain variable region comprising the amino acidsequence DIQMTQSPSSLSASVGDRVTITCKASQDINSYLTWFQQKPGKAPKSLIYLTNRLMDGVPSRFSGSGSGTDFFLTISSLQPEDFATYYCLQYVDFPYTFGQ GTKVEIKorDIKMTQSPSSLSASVGDRVTITCKASQDINSYLTWFQQKPGKAPKTLIYLTNRLMDGVPSRFSGSGSGQEFLLTISSLQPEDFATYYCLQ YVDFPYTFGQGTKVEIK;and/or ii) at least one heavy chain variable region comprising the aminoacid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSSSAMSWVRQAPGKGLEWVSTISSGGRSTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKLI SPYWGQGTLVTVSS orEVQLLESGGGLVQPGGSLRLSCAASGIIFSSSAMSWVRQAPGKGLEWVATISSGGRSTYYPDSVKGRFTVSRDSSKNTLYLQMNSLRAEDTAVYYCAKLI SPYWGQGTLVTVSS.These sequences correspond to humanised variants of antibody BB-7 (seeTables 23, 24 and 24A).

In an embodiment of any aspect of the present invention the antibody orantigen-binding fragment thereof may comprise one or more of thefollowing amino acid sequences:

KASQDINSYLT; and/or LVNRLVD; and/or LQYDDFPYT and/or THAMS; and/orTISSGGRSTYYPDSVKG; and/or LISTY.

The sequences of the immediately preceding embodiment are considered tocomprise the complementarity determining regions of the light and heavyvariable regions of the exemplary antibody DC-1 (see FIG. 20). In afurther embodiment, the antibody may comprise the amino acid sequenceKASQDINSYLTXXXXXXXXXXXXXXXLVNRLVDXXXXXXXXXXXXXXXAXXXXXXXXXXXXXXXXLQYDDFPYT or KASQDINSYLTXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXA; orLVNRLVDXXXXXXXXXXXXXXXA; or AXXXXXXXXXXXXXXXXLQYDDFPYT, wherein X is anyamino acid.

Thus, in an embodiment of any aspect of the present invention, theantibody or antigen-binding fragment thereof may have at least one lightchain variable region comprising the following sequences: KASQDINSYLT;and LVNRLVD; and LQYDDFPYT

In a further embodiment of any aspect of the present invention, theantibody or antigen-binding fragment thereof may have at least one heavychain variable region comprising the following sequences: THAMS; andTISSGGRSTYYPDSVKG; and LISTY.

In an embodiment of any aspect of the present invention the antibody orantigen-binding fragment thereof may have at least one light chainvariable region comprising the amino acid sequenceDITMTQSPSSIYASLGERVTITCKASQDINSYLTWFQQKPGKSPKILIYLVNRLVDGVPSRFSGSGSGQDYALTISSLEYEDMGIYYCLQYDDFPYTFGGGTKLEIK. This light chainvariable region corresponds with that found in exemplary antibody DC-1(FIG. 20).

In an embodiment of any aspect of the present invention, the antibody orantigen-binding fragment thereof may have at least one heavy chainvariable region comprising the amino acid sequenceEVQLVESGGGLVKPGGSLKLSCAASGFTLSTHAMSWVRQTPEKRLEWVATISSGGRSTYYPDSVKGRFTISRDNVKNTLYLQLSSLRSEDTAVYFCARLISTYWGQGTTLTVSS. This heavy chainvariable region corresponds with that found in exemplary antibody DC-1(FIG. 20).

Thus, it is envisaged that in an embodiment of any aspect of the presentinvention, the antibody or antigen-binding fragment thereof may have:

i) at least one light chain variable region comprising the amino acidsequence DITMTQSPSSIYASLGERVTITCKASQDINSYLTWFQQKPGKSPKILIYLVNRLVDGVPSRFSGSGSGQDYALTISSLEYEDMGIYYCLQYDDFPYTFGG GTKLEIK; and ii) atleast one heavy chain variable region comprising the amino acid sequenceEVQLVESGGGLVKPGGSLKLSCAASGFTLSTHAMSWVRQTPEKRLEWVATISSGGRSTYYPDSVKGRFTISRDNVKNTLYLQLSSLRSEDTAVYFCARLI STYWGQGTTLTVS.

It is further envisaged that in an embodiment of any aspect of thepresent invention, the antibody or antigen-binding fragment thereof mayhave:

i) at least one light chain variable region comprising the amino acidsequence DIQMTQSPSSLSASVGDRVTITCKASQDINSYLTWFQQKPGKAPKSLIYLVNRLVDGVPSRFSGSGSGTDFFLTISSLQPEDFATYYCLQYDDFPYTFGQ GTKVEIK orDITMTQSPSSLSASVGDRVTITCKASQDINSYLTWFQQKPGKAPKILIYLVNRLVDGVPSRFSGSGSGQDYALTISSLQPEDFATYYCLQYDDFPYTFGQ GTKVEIK; and/or ii)at least one heavy chain variable region comprising the amino acidsequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSTHAMSWVRQAPGKGLEWVSTISSGGRSTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKLI STYWGQGTLVTVSS orEVQLLESGGGLVQPGGSLRLSCAASGFTLSTHAMSWVRQAPGKGLEWVATISSGGRSTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCARLI STYWGQGTLVTVSS.These sequences correspond with the humanised variants of DC-1 providedin Tables 23, 24 and 24A.

In an embodiment of any aspect of the present invention the antibody orantigen-binding fragment thereof may comprise one or more of thefollowing amino acid sequences:

KASQDINSYLT; and/or XXNRLXD; and/or LQYXDFPYT; and/or XXAMS; and/orTISXGGXXTYYPDSVKG; and/or LISXY, wherein X is any amino acid.

In an embodiment of any aspect of the present invention the antibody orantigen-binding fragment thereof may comprise one or more of thefollowing amino acid sequences:

(K/Q/R)ASQ(D/G)I(N/S/R)(S/N)YL(T/N/A); and/or(R/L/V/D/A)(T/V/A)(N/S)(R/N)L(F/M/V/E/Q)(D/T/S); and/or(L/Q)Q(Y/H)(D/V/N)(D/T)(F/Y)P(Y/L/W)T; and/or (S/T)(S/H/Y)AMS; and/or(T/A)IS(V/S/G)(G/S)G(G/R)(K/S)TYY(P/A)DSVKG; and/or(L/D)(I/G)(S/G)(L/P/T/V)Y.

In a further embodiment of any aspect of the present invention theantibody or antigen-binding fragment thereof may have at least one lightchain variable region comprising the amino acid sequenceQIVLTQSPAIMSASPGEKVTMTCSASSSVDYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMGAEDAATYYCQQWNSSPLTFGAGTKLELK. This light chainvariable region corresponds with that found in exemplary antibody DD-9(FIG. 25).

In an embodiment of any aspect of the present invention, the antibody orantigen-binding fragment thereof may have at least one heavy chainvariable region comprising the amino acid sequenceQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVSWIRQSSGKGLEWLAHIYWDDDKRYNPSLKSRITISKDSSSNQVFLKITSVDTADTATYYCARSWTTAPFAFWGQGTLVTVSA. This heavychain variable region corresponds with that found in exemplary antibodyDD-9 (FIG. 25).

Thus, it is envisaged that in an embodiment of any aspect of the presentinvention, the antibody or antigen-binding fragment thereof may have:

i) at least one light chain variable region comprising the amino acidsequence QIVLTQSPAIMSASPGEKVTMTCSASSSVDYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMGAEDAATYYCQQWNSSPLTFGAG TKLELK; and ii) atleast one heavy chain variable region comprising the amino acid sequenceQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVSWIRQSSGKGLEWLAHIYWDDDKRYNPSLKSRITISKDSSSNQVFLKITSVDTADTATYYCARS WTTAPFAFWGQGTLVTVSA.

In a further embodiment of any aspect of the present invention theantibody or antigen-binding fragment thereof may have at least one lightchain variable region comprising the amino acid sequenceQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATFYCQQWSSSPLTFGAGTKLELK. This light chainvariable region corresponds with that found in exemplary antibody DH-2(FIG. 26).

In an embodiment of any aspect of the present invention, the antibody orantigen-binding fragment thereof may have at least one heavy chainvariable region comprising the amino acid sequenceQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWLAHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITSVDTADTATYYCARSGTTAPFAYWGQGTLVTVSA. This heavychain variable region corresponds with that found in exemplary antibodyDH-2 (FIG. 26).

Thus, it is envisaged that in an embodiment of any aspect of the presentinvention, the antibody or antigen-binding fragment thereof may have:

i) at least one light chain variable region comprising the amino acidsequence QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATFYCQQWSSSPLTFGAG TKLELK; and ii) atleast one heavy chain variable region comprising the amino acid sequenceQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWLAHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITSVDTADTATYYCARS GTTAPFAYWGQGTLVTVSA.

In an embodiment of any aspect of the invention, it is envisaged that,the antibody or antigen-binding fragment thereof may have:

-   -   i) at least one light chain variable region comprising an amino        acid sequence corresponding with any of the VK sequences        provided in any of FIGS. 18 to 28; and/or    -   ii) at least one heavy chain variable region comprising an amino        acid sequence corresponding with any of the VH sequences        provided in any of FIGS. 18 to 28 or a fragment, variant or        derivative thereof.

As indicated above, it is envisaged that the antibody or antigen-bindingfragment of the preceding embodiments may comprise an amino acidsequence having at least 75% identity with one or more of the amino acidsequences given above, for example at least 80%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% identitywith one or more of the amino acid sequences specified above. It is alsoenvisaged that the antibody or antigen-binding fragment may comprise upto 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more insertions, deletions,conservative substitutions and/or non-conservative substitutions.

In a second aspect, the invention provides an antibody orantigen-binding fragment thereof comprising one or more of the followingamino acid sequences:

KASQDINSYLT; and/or XXNRLXD; and/or LQYXDFPYT; and/or XXAMS; and/orTISXGGXXTYYPDSVKG; and/or LISXY, wherein X is any amino acid.

In an embodiment, the invention provides an antibody or antigen-bindingfragment thereof comprising one or more of the following amino acidsequences:

(K/Q/R)ASQ(D/G)I(N/S/R)(S/N)YL(T/N/A); and/or(R/L/V/D/A)(T/V/A)(N/S)(R/N)L(F/M/V/E/Q)(D/T/S); and/or(L/Q)Q(Y/H)(D/V/N)(D/T)(F/Y)P(Y/L/W)T; and/or (S/T)(S/H/Y)AMS; and/or(T/A)IS(V/S/G)(G/S)G(G/R)(K/S)TYY(P/A)DSVKG; and/or(L/D)(I/G)(S/G)(L/P/T/V)Y.

In an embodiment, the invention provides an antibody or antigen-bindingfragment thereof comprising one or more of the following amino acidsequences:

KASQDINSYLT; and/or RTNRLFD; and/or LQYDDFPYT; and/or SSAMS; and/orTISVGGGKTYYPDSVKG; and/or LISLY.

In an embodiment, the antibody or antigen-binding fragment thereof ofthe second aspect may comprise one or more of the following amino acidsequences:

TCKASQDINSYLTWF; and/or TLIYRTNRLFDGVP or TLIYRTNRLFDGVPXXFSGSGSGQDFF;and/or YCLQYDDFPYTFG; and/or FTLSSSAMSWVR or CXAXXFTLSSSAMSWVR; and/orWVATISVGGGKTYYPDSVKGRFTISR or WVATISVGGGKTYYPDSVKGRFTISRXNXXXXL; and/orYCAKLISLYVVG, wherein X is any amino acid.

The sequences of the preceding embodiment are considered to comprise thecomplementarity determining regions of the light and heavy variableregions of the exemplified antibody AB-1.

In a further embodiment, the antibody or antigen-binding fragmentthereof may comprise the amino acid sequenceKASQDINSYLTXXXXXXXXXXXXXXXRTNRLFDXXXXXXXXXXXXXXXFXXXXXXXXXXXXXXXXLQYDDFPYT; or KASQDINSYLTXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXF;or RTNRLFDXXXXXXXXXXXXXXXF; or FXXXXXXXXXXXXXXXXLQYDDFPYT, wherein X isany amino acid.

In a further embodiment, the antibody or antigen-binding fragmentthereof may comprise the amino acid sequence TCKASQDINSYLTWF orTCKASQDINSYLTWY; and/or LLIYRTNRLFDGVP or SLIYRTNRLFDGVP orLLIYRTNRLFDGVPXXFSGSGSGQDFF or SLIYRTNRLFDGVPXXFSGSGSGQDFF; and/orYCLQYDDFPYTFG; and/or FTFSSSAMSWVR or CXAXXFTFSSSAMSWVR; and/orWVSTISVGGGKTYYPDSVKGRFTISR or WVSTISVGGGKTYYPDSVKGRFTISRXNXXXXL; and/orYCAKLISLYVVG, wherein X is any amino acid.

Thus, in an embodiment, the antibody or antigen-binding fragment thereofmay have at least one light chain variable region comprising thefollowing CDRs: KASQDINSYLT; and RTNRLFD; and LQYDDFPYT.

In a further embodiment, the antibody or antigen-binding fragmentthereof may have at least one heavy chain variable region comprising thefollowing CDRs: SSAMS; and TISVGGGKTYYPDSVKG; and LISLY.

In a further embodiment, the antibody or antigen-binding fragmentthereof of the second aspect may have at least one light chain variableregion comprising the amino acid sequence

DIQMTQTPSSMYASLGERVTITCKASQDINSYLTWFQQKPGKSPKTLIYRTNRLFDGVPSRFSGSGSGQDFFLTISSLEYEDMGIYYCLQYDDFPYTFGG GTKLEIK orDIKMTQSPSSMYASLGERVTITCKASQDINSYLTWFQQKPGKSPKTLIYRTNRLFDGVPSRFSGSGSGQDFFLTISSLEYEDMGIYYCLQYDDFPYTFGG GTKLEIK, orEIVLTQSPSSMYASLGERVTITCKASQDINSYLTWFQQKPGKSPKTLIYRTNRLFDGVPSRFSGSGSGQDFFLTISSLEYEDMGIYYCLQYDDFPYTFGG GTKLEIK (AB-1_VK), orDIQMTQSPSSMYASLGERVTITCKASQDINSYLTWFQQKPGKSPKTLIYRTNRLFDGVPSRFSGSGSGQDFFLTISSLEYEDMGIYYCLQYDDFPYTFGG GTKLEIK (AB-1_VK1),or EIVLTQSPSSLSASVGDRVTITCKASQDINSYLTWYQQKPGKAPKLLIYRTNRLFDGVPSRFSGSGSGTDFFFTISSLQPEDFGTYYCLQYDDFPYTFGG GTKLEIK (hAB-1_RKE),or DIQMTQSPSSLSASVGDRVTITCKASQDINSYLTWFQQKPGKAPKSLIYRTNRLFDGVPSRFSGSGSGTDFFLTISSLQPEDFATYYCLQYDDFPYTFGQ GTKVEIK (hAB-1_RKJ).

In a yet further embodiment of the second aspect, the antibody orantigen-binding fragment thereof may have at least one heavy chainvariable region comprising the amino acid sequence

EVQLEESGGGLVKPGGSLKLSCAASGFTLSSSAMSWVRQTPDRRLEWVATISVGGGKTYYPDSVKGRFTISRDNAKNTLYLQMNSLRSEDTAMYYCAKLI SLYWGQGTTLTVSS orEVQLVESGGGLVKPGGSLKLSCAASGFTLSSSAMSWVRQTPDRRLEWVATISVGGGKTYYPDSVKGRFTISRDNAKNTLYLQMNSLRSEDTAMYYCAKLI SLYWGQGTTLTVSS, orEVQLQESGGGLVKPGGSLKLSCAASGFTLSSSAMSWVRQTPDRRLEWVATISVGGGKTYYPDSVKGRFTISRDNAKNTLYLQMNSLRSEDTAMYYCAKLI SLYWGQGTTLTVSS(AB-1_VH), or EVQLLESGGGLVQPGGSLRLSCAASGFTFSSSAMSWVRQAPGKGLEWVSTISVGGGKTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKLI SLYWGQGTLVTVSS(hAB-1_RHA).

The invention also provides an antibody or antigen-binding fragmentthereof having at least one light chain variable region as embodied inthe second aspect and at least one heavy chain variable region asembodied in the second aspect.

For the avoidance of doubt, the antibody or antigen-binding fragmentthereof of the second aspect may comprise any amino acid sequenceprovided in relation to the first aspect. Thus, the antibody orantigen-binding fragment thereof of the second aspect may include any VKand/or VK region as exemplified in any of FIGS. 18 to 28 or Tables 14 to24 and 24A, or any variant, fragment or derivative thereof.

Further, the antibody or antigen-binding fragment thereof of any aspectof the invention may be or comprise any humanised or chimeric antibodydescribed herein, in particular, antibody or antigen-binding fragmentthereof may comprise or consist of any of the sequences provided inTables 14 to 24 and 24A.

It is envisaged that the antibody or antigen-binding fragment of thesecond aspect and related embodiments may comprise an amino acidsequence having at least 75% identity with one or more of the amino acidsequences given above, for example at least 80%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% identitywith one or more of the amino acid sequences specified above. It is alsoenvisaged that the antibody or antigen-binding fragment may comprise upto 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more insertions, deletions,conservative substitutions and/or non-conservative substitutions.

In a preferred embodiment of any aspect of the invention, the antibodyor antigen-binding fragment thereof may comprise or consist of an intactantibody. Alternatively, the antibody or antigen-binding fragmentthereof may consist essentially of an intact antibody. By “consistessentially of” we mean that the antibody or antigen-binding fragmentthereof consists of a portion of an intact antibody sufficient todisplay binding specificity for TG2.

The antibody or antigen-binding fragment of the invention may be anon-naturally occurring antibody. Of course, where the antibody is anaturally occurring antibody, it is provided in an isolated form (i.e.distinct from that in which it is found in nature).

In an embodiment of any aspect of the invention, the antibody orantigen-binding fragment thereof may comprise or consist of anantigen-binding fragment selected from the group consisting of: an Fvfragment; an Fab fragment; and an Fab-like fragment. In a furtherembodiment, the Fv fragment may be a single chain Fv fragment or adisulphide-bonded Fv fragment. In a yet further embodiment, the Fab-likefragment may be an Fab′ fragment or an F(ab)₂ fragment.

The variable heavy (V_(H)) and variable light (V_(L)) domains of theantibody are involved in antigen recognition, a fact first recognised byearly protease digestion experiments. Further confirmation was found by“humanisation” of rodent antibodies. Variable domains of rodent originmay be fused to constant domains of human origin such that the resultantantibody retains the antigenic specificity of the rodent-parentedantibody (Morrison et al (1984) Proc. Natl. Acad. Sci. USA 81,6851-6855).

Antigenic specificity is conferred by variable domains and isindependent of the constant domains, as known from experiments involvingthe bacterial expression of antibody fragments, all containing one ormore variable domains. These molecules include Fab-like molecules(Better et al (1988) Science 240, 1041); Fv molecules (Skerra et al(1988) Science 240, 1038); single-chain Fv (ScFv) molecules where theV_(H) and V_(L) partner domains are linked via a flexible oligopeptide(Bird et al (1988) Science 242, 423; Huston et al (1988) Proc. Natl.Acad. Sci. USA 85, 5879) and single domain antibodies (dAbs) comprisingor consisting of isolated V domains (Ward et al (1989) Nature 341, 544).A general review of the techniques involved in the synthesis of antibodyfragments which retain their specific binding sites is to be found inWinter & Milstein (1991) Nature 349, 293-299.

Thus, by “antigen-binding fragment” we include a functional fragment ofan antibody that is capable of binding to TG2.

Exemplary antigen-binding fragments may be selected from the groupconsisting of Fv fragments (e.g. single chain Fv and disulphide-bondedFv), and Fab-like fragments (e.g. Fab fragments, Fab′ fragments andF(ab)₂ fragments).

In one embodiment of the invention, the antigen-binding fragment is anscFv.

The advantages of using antibody fragments, rather than wholeantibodies, are several-fold. The smaller size of the fragments may leadto improved pharmacological properties, such as better penetration ofsolid tissue. Moreover, antigen-binding fragments such as Fab, Fv, ScFvand dAb antibody fragments can be expressed in and secreted from E.coli, thus allowing the facile production of large amounts of the saidfragments.

Also included within the scope of the invention are modified versions ofantibodies and an antigen-binding fragments thereof, e.g. modified bythe covalent attachment of polyethylene glycol or other suitablepolymer.

It is particularly preferred that the antibody or antigen-bindingfragment thereof is a recombinant molecule.

Methods of generating antibodies and antibody fragments are well knownin the art. For example, antibodies may be generated via any one ofseveral methods which employ induction of in vivo production of antibodymolecules, screening of immunoglobulin libraries (Orlandi. et al, 1989.Proc. Natl. Acad. Sci. U.S.A. 86:3833-3837; Winter et al., 1991, Nature349:293-299) or generation of monoclonal antibody molecules by celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the Epstein-Barrvirus (EBV)-hybridoma technique (Kohler et al., 1975. Nature256:4950497; Kozbor et al., 1985. J. Immunol. Methods 81:31-42; Cote etal., 1983. Proc. Natl. Acad. Sci. USA 80:2026-2030; Cole et al., 1984.Mol. Cell. Biol. 62:109-120).

Conveniently, the invention provides an antibody or antigen-bindingfragment thereof wherein the antibody is a recombinant antibody (i.e.wherein it is produced by recombinant means).

In a particularly preferred embodiment of any aspect of the invention,the antibody may be a monoclonal antibody.

Suitable monoclonal antibodies to selected antigens may be prepared byknown techniques, for example those disclosed in “Monoclonal Antibodies:A manual of techniques”, H Zola (CRC Press, 1988) and in “MonoclonalHybridoma Antibodies: Techniques and Applications”, J G R Hurrell (CRCPress, 1982), which are incorporated herein by reference. Exemplarymonoclonal antibodies of the invention and suitable methods for theirmanufacture are provided in the Examples below.

Antibody fragments can also be obtained using methods well known in theart (see, for example, Harlow & Lane, 1988, “Antibodies: A LaboratoryManual”, Cold Spring Harbor Laboratory, New York, which is incorporatedherein by reference). For example, antibody fragments of the presentinvention can be prepared by proteolytic hydrolysis of the antibody orby expression in E. coli or mammalian cells (e.g. Chinese hamster ovarycell culture or other protein expression systems) of DNA encoding thefragment. Alternatively, antibody fragments can be obtained by pepsin orpapain digestion of whole antibodies by conventional methods.

In an embodiment of any aspect of the invention, the antibody orantigen-binding fragment thereof may be a human antibody or a humanisedantibody.

It will be appreciated by persons skilled in the art that for humantherapy or diagnostics, humanised antibodies may be used. Humanisedforms of non-human (e.g. murine) antibodies are genetically engineeredchimeric antibodies or antibody fragments having minimal-portionsderived from non-human antibodies. Humanised antibodies includeantibodies in which complementary determining regions of a humanantibody (recipient antibody) are replaced by residues from acomplementary determining region of a non human species (donor antibody)such as mouse, rat or rabbit having the desired functionality. In someinstances, Fv framework residues of the human antibody are replaced bycorresponding non-human residues. Humanised antibodies may also compriseresidues which are found neither in the recipient antibody nor in theimported complementarity determining region or framework sequences. Ingeneral, the humanised antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the complementarity determining regions correspondto those of a non-human antibody and all, or substantially all, of theframework regions correspond to those of a relevant human consensussequence. Humanised antibodies optimally also include at least a portionof an antibody constant region, such as an Fc region, typically derivedfrom a human antibody (see, for example, Jones et al., 1986. Nature321:522-525; Riechmann et al., 1988, Nature 332:323-329; Presta, 1992,Curr. Op. Struct. Biol. 2:593-596, which are incorporated herein byreference).

Methods for humanising non-human antibodies are well known in the art.Generally, the humanised antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues, often referred to as imported residues, aretypically taken from an imported variable domain. Humanisation can beessentially performed as described (see, for example, Jones et al.,1986, Nature 321:522-525; Reichmann et al., 1988. Nature 332:323-327;Verhoeyen et al., 1988, Science 239:1534-15361; U.S. Pat. No. 4,816,567,which are incorporated herein by reference) by substituting humancomplementarity determining regions with corresponding rodentcomplementarity determining regions. Accordingly, such humanisedantibodies are chimeric antibodies, wherein substantially less than anintact human variable domain has been substituted by the correspondingsequence from a non-human species. In practice, humanised antibodies maybe typically human antibodies in which some complementarity determiningregion residues and possibly some framework residues are substituted byresidues from analogous sites in rodent antibodies.

Human antibodies can also be identified using various techniques knownin the art, including phage display libraries (see, for example,Hoogenboom & Winter, 1991, J. Mol. Biol. 227:381; Marks et al., 1991, J.Mol. Biol. 222:581; Cole et al., 1985, In: Monoclonal antibodies andCancer Therapy, Alan R. Liss, pp. 77; Boerner et al., 1991. J. Immunol.147:86-95, Soderlind et al., 2000, Nat Biotechnol 18:852-6 and WO98/32845 which are incorporated herein by reference).

The term “amino acid” as used herein includes the standard twentygenetically-encoded amino acids and their corresponding stereoisomers inthe ‘D’ form (as compared to the natural ‘L’ form), omega-amino acidsother naturally-occurring amino acids, unconventional amino acids (e.g.α,α-disubstituted amino acids, N-alkyl amino acids, etc.) and chemicallyderivatised amino acids.

When an amino acid is being specifically enumerated, such as “alanine”or “Ala” or “A”, the term refers to both L-alanine and D-alanine unlessexplicitly stated otherwise. Other unconventional amino acids may alsobe suitable components for polypeptides of the present invention, aslong as the desired functional property is retained by the polypeptide.For the peptides shown, each encoded amino acid residue, whereappropriate, is represented by a single letter designation,corresponding to the trivial name of the conventional amino acid.

In one embodiment, the polypeptides as defined herein comprise orconsist of L-amino acids.

Once suitable antibodies are obtained, they may be tested for activity,such as binding specificity or a biological activity of the antibody,for example by ELISA, immunohistochemistry, flow cytometry,immunoprecipitation, Western blots, etc. The biological activity may betested in different assays with readouts for that particular feature.

In a further aspect, the present invention provides a polynucleotideencoding an antibody or an antigen-binding fragment thereof according tothe second aspect and the related embodiments of the second aspect.

Accordingly, in an embodiment, the invention provides an isolatedpolynucleotide comprising or consisting of the nucleotide sequences:

i) GACATCCAGATGACACAGACTCCATCTTCCATGTATGCATCTCTAGGAGAGAGAGTCACTATCACTTGCAAGGCGAGTCAGGACATTAATAGCTATTTAACCTGGTTCCAGCAGAAACCAGGGAAATCTCCTAAGACCCTGATCTATCGTACAAATAGATTGTTTGATGGGGTCCCATCCAGGTTCAGTGGCAGTGGATCTGGGCAAGATTTTTTTCTCACCATCAGCAGCCTGGAATATGAAGATATGGGAATTTATTATTGTCTACAGTATGATGACTTTCCGTACACGTTCGGAGGGGGGACCAAACTGGAAATAAAA; and/or ii)GAAGTACAGCTGGAGGAGTCAGGGGGGGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTCTCAGTTCCTCTGCCATGTCTTGGGTTCGCCAGACTCCGGACAGGAGGCTGGAGTGGGTCGCAACCATTAGTGTTGGTGGTGGTAAAACCTACTATCCAGACAGTGTGAAGGGTCGCTTCACCATCTCCAGAGACAATGCCAAGAACACCCTCTATCTGCAAATGAACAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGCAAAACTAATCAGTCTCTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA.

In a further embodiment, the invention provides an isolatedpolynucleotide comprising or consisting of any of the nucleic acidsequences listed in any of FIGS. 18 to 28.

Thus, in an embodiment, the invention provides an isolatedpolynucleotide comprising or consisting of the nucleotide sequences:

i) GCCATCAAGATGACCCAGTCTCCATCTTCCATGTATGCATCTCTAGGAGAGAGAGTCATCATCACTTGCAAGGCGAGTCAGGACATAAATAGTTATTTAACCTGGTTCCAACAGAAACCAGGAAAGTCTCCTAAGACCCTGATCTATCTTACAAATAGATTGATGGATGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGCAAGAATTTTTACTCACCATCAGCGGCCTGGAACATGAAGATATGGGCATTTATTATTGTCTCCAGTATGTTGACTTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA; and/or ii)GCAGTGCAACTGGTAGAGTCTGGGGGAGGCTTGGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGAATCATTTTCAGTTCCTCTGCCATGTCTTGGGTTCGCCAGACTCCGGAAAAGAGACTGGAGTGGGTCGCAACTATTAGTAGTGGTGGTCGTTCCACCTACTATCCAGACAGTGTGAAGGGTCGATTCACCGTCTCCAGAGACAGTGCCAAGAACACCCTATACCTGCAAATGGACAGTCTGAGGTCTGAGGACACGGCCATTTATTACTGTGCAAAACTAATCAGTCCCTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA.

In a further embodiment, the invention provides an isolatedpolynucleotide comprising or consisting of the nucleotide sequences:

i) GACATCACGATGACCCAGTCTCCATCTTCCATATATGCATCTCTGGGAGAGAGAGTCACTATCACTTGCAAGGCGAGTCAGGACATTAATAGCTATTTAACCTGGTTCCAGCAGAAACCAGGGAAATCTCCTAAGATCCTGATCTATCTTGTAAATAGATTGGTAGATGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGCAAGATTATGCTCTCACCATCAGCAGTCTGGAATATGAAGATATGGGAATTTATTATTGTCTACAATATGATGACTTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA; and/or ii)GAAGTGCAGTTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTCTCAGTACCCATGCCATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGAGTGGGTCGCAACCATTAGTAGTGGTGGTCGTTCCACCTACTATCCAGACAGTGTGAAGGGTCGATTCACTATCTCCAGAGACAATGTCAAGAACACCCTATATCTGCAACTGAGCAGTCTGAGGTCTGAGGACACGGCCGTGTATTTCTGTGCAAGACTAATCAGTACCTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA.

It is preferred that the antibody or antigen-binding fragment thereof ofthe invention inhibits TG2 activity. Thus, it is preferred that theantibody of the invention inhibits TG2 enzymatic activity and thusprevents it from functioning to cross-link lysine and glutamine residueswith Nϵ-(γ-glutamyl)lysine isopeptide bonds. It is preferred that theenzymatic activity of TG2 is completely abrogated, but it is envisagedthat the inhibition may be partial inhibition in instances where thispartial inhibition is sufficient to provide a useful therapeutic ornon-therapeutic outcome. The skilled person would be able to determinewhether the extent of inhibition is effective to achieve the desiredoutcome.

It is preferred that the antibody or antigen-binding fragment thereof isspecific for TG2 inhibition. Thus, it is preferred that the antibody orantigen-binding fragment thereof does not inhibit TG1, TG3, TG13 and/orTG7 activity. It is envisaged that an antibody that effectively inhibitsTG2 activity but is sufficiently selective so that it does notsignificantly inhibit TG1, TG3, TG13 and/or TG7 activity may beparticularly useful in medicine. Thus, it is preferred that the antibodyexclusively inhibits TG2 activity.

In a further aspect, the invention provides an antibody orantigen-binding fragment thereof whose binding to TG2 (for example,human TG2) is inhibited or reduced when an antibody according to anypreceding aspect is bound to TG2 (for example, human TG2).

Thus, the invention includes any antibody that selectively binds to anepitope within the region of TG2 such that it may compete with anddisrupt binding of any antibody of the preceding aspects.

In a further aspect, the present invention provides a compoundcomprising an antibody or antigen-binding fragment thereof according toany preceding aspect and a further moiety.

In an embodiment, the further moiety may be a directly or indirectlycytotoxic moiety.

By “directly cytotoxic” we include the meaning that the moiety is onewhich on its own is cytotoxic. By “indirectly cytotoxic” we include themeaning that the moiety is one which, although is not itself cytotoxic,can induce cytotoxicity, for example by its action on a further moleculeor by further action on it.

The cytotoxic moiety may be selected from, but is not limited to, thegroup comprising a directly cytotoxic chemotherapeutic agent, a directlycytotoxic polypeptide, a moiety which is able to convert a relativelynon-toxic prodrug into a cytotoxic drug, a radiosensitizer, a directlycytotoxic nucleic acid, a nucleic acid molecule that encodes a directlyor indirectly cytotoxic polypeptide, a nucleic acid molecule thatencodes a therapeutic polypeptide, or a radioactive atom. It isenvisaged that the radioactive atom may be any one of phosphorus-32,iodine-125, iodine-131, indium-111, rhenium-186, rhenium-188 oryttrium-90.

Cytotoxic chemotherapeutic agents, such as anticancer agents, include:alkylating agents including nitrogen mustards such as mechlorethamine(HN₂), cyclophosphamide, ifosfamide, melphalan (L-sarcolysin) andchlorambucil; ethylenimines and methylmelamines such ashexamethylmelamine, thiotepa; alkyl sulphonates such as busulfan;nitrosoureas such as carmustine (BCNU), lomustine (CCNU), semustine(methyl-CCNU) and streptozocin (streptozotocin); and triazenes such asdecarbazine (DTIC; dimethyltriazenoimidazole-carboxamide);Antimetabolites including folic acid analogues such as methotrexate(amethopterin); pyrimidine analogues such as fluorouracil(5-fluorouracil; 5-FU), floxuridine (fluorodeoxyuridine; FUdR) andcytarabine (cytosine arabinoside); and purine analogues and relatedinhibitors such as mercaptopurine (6-mercaptopurine; 6-MP), thioguanine(6-thioguanine; TG) and pentostatin (2′-deoxycoformycin). NaturalProducts including vinca alkaloids such as vinblastine (VLB) andvincristine; epipodophyllotoxins such as etoposide and teniposide;antibiotics such as dactinomycin (actinomycin D), daunorubicin(daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin(mithramycin) and mitomycin (mitomycin C); enzymes such asL-asparaginase; and biological response modifiers such as interferonalphenomes. Miscellaneous agents including platinum coordinationcomplexes such as cisplatin (cis-DDP) and carboplatin; anthracenedionesuch as mitoxantrone and anthracycline; substituted urea such ashydroxyurea; methyl hydrazine derivative such as procarbazine(N-methylhydrazine, MIH); and adrenocortical suppressant such asmitotane (o,p′-DDD) and aminoglutethimide; taxol andanalogues/derivatives; and hormone agonists/antagonists such asflutamide and tamoxifen.

Various of these agents have previously been attached to antibodies andother target site-delivery agents, and so compounds of the inventioncomprising these agents may readily be made by the person skilled in theart. For example, carbodiimide conjugation (Bauminger & Wilchek (1980)Methods Enzymol. 70, 151-159; incorporated herein by reference) may beused to conjugate a variety of agents, including doxorubicin, toantibodies.

Cytotoxic peptides or polypeptide moieties include any moiety whichleads to cell death. Cytotoxic peptide and polypeptide moieties are wellknown in the art and include, for example, ricin, abrin, Pseudomonasexotoxin, tissue factor and the like. Methods for linking them totargeting moieties such as antibodies are also known in the art. The useof ricin as a cytotoxic agent is described in Burrows & Thorpe (1993)Proc. Natl. Acad. Sci. USA 90, 8996-9000, incorporated herein byreference, and the use of tissue factor, which leads to localised bloodclotting and infarction of a tumour, has been described by Ran et al(1998) Cancer Res. 58, 4646-4653 and Huang et al (1997) Science 275,547-550. Tsai et al (1995) Dis. Colon Rectum 38, 1067-1074 describes theabrin A chain conjugated to a monoclonal antibody and is incorporatedherein by reference. Other ribosome inactivating proteins are describedas cytotoxic agents in WO 96/06641. Pseudomonas exotoxin may also beused as the cytotoxic polypeptide moiety (see, for example, Aiello et al(1995) Proc. Natl. Acad. Sci. USA 92, 10457-10461; incorporated hereinby reference).

Certain cytokines, such as TNFα and IL-2, may also be useful ascytotoxic agents.

Certain radioactive atoms may also be cytotoxic if delivered insufficient doses. Thus, the cytotoxic moiety may comprise a radioactiveatom which, in use, delivers a sufficient quantity of radioactivity tothe target site so as to be cytotoxic. Suitable radioactive atomsinclude phosphorus-32, iodine-125, iodine-131, indium-111, rhenium-186,rhenium-188 or yttrium-90, or any other isotope which emits enoughenergy to destroy neighbouring cells, organelles or nucleic acid.Preferably, the isotopes and density of radioactive atoms in thecompound of the invention are such that a dose of more than 4000 cGy(preferably at least 6000, 8000 or 10000 cGy) is delivered to the targetsite and, preferably, to the cells at the target site and theirorganelles, particularly the nucleus. The radioactive atom may beattached to the antibody in known ways. For example EDTA or anotherchelating agent may be attached to the antibody and used to attach ¹¹¹Inor ⁹⁰Y. Tyrosine residues may be labelled with ¹²⁵I or ¹³¹I.

The cytotoxic moiety may be a suitable indirectly cytotoxic polypeptide.In a particularly preferred embodiment, the indirectly cytotoxicpolypeptide is a polypeptide which has enzymatic activity and canconvert a relatively non-toxic prodrug into a cytotoxic drug. When thetargeting moiety is an antibody this type of system is often referred toas ADEPT (Antibody-Directed Enzyme Prodrug Therapy). The system requiresthat the targeting moiety locates the enzymatic portion to the desiredsite in the body of the patient (i.e. the site expressing MR, such asnew vascular tissue associated with a tumour) and after allowing timefor the enzyme to localise at the site, administering a prodrug which isa substrate for the enzyme, the end product of the catalysis being acytotoxic compound. The object of the approach is to maximise theconcentration of drug at the desired site and to minimise theconcentration of drug in normal tissues (see Senter, P. D. et al (1988)“Anti-tumor effects of antibody-alkaline phosphatase conjugates incombination with etoposide phosphate” Proc. Natl. Acad. Sci. USA 85,4842-4846; Bagshawe (1987) Br. J. Cancer 56, 531-2; and Bagshawe, K. D.et al (1988) “A cytotoxic agent can be generated selectively at cancersites” Br. J. Cancer. 58, 700-703.)

The cytotoxic substance may be any existing anti-cancer drug such as analkylating agent; an agent which intercalates in DNA; an agent whichinhibits any key enzymes such as dihydrofolate reductase, thymidinesynthetase, ribonucleotide reductase, nucleoside kinases ortopoisomerase; or an agent which effects cell death by interacting withany other cellular constituent. Etoposide is an example of atopoisomerase inhibitor.

Reported prodrug systems include: a phenol mustard prodrug activated byan E. coli β-glucuronidase (Wang et al, 1992 and Roffler et al, 1991); adoxorubicin prodrug activated by a human β-glucuronidase (Bosslet et al,1994); further doxorubicin prodrugs activated by coffee beanα-galactosidase (Azoulay et al, 1995); daunorubicin prodrugs, activatedby coffee bean α-D-galactosidase (Gesson et al, 1994); a 5-fluorouridineprodrug activated by an E. coli β-D-galactosidase (Abraham et al, 1994);and methotrexate prodrugs (e.g. methotrexate-alanine) activated bycarboxypeptidase A (Kuefner et al, 1990, Vitols et al, 1992 and Vitolset al, 1995). These and others are included in Table 1.

TABLE 1 Enzyme Prodrug Carboxypeptidase G2 Derivatives of L-glutamicacid and benzoic acid mustards, aniline mustards, phenol mustards andphenylenediamine mustards; fluorinated derivatives of these Alkalinephosphatase Etoposide phosphate Mitomycin phosphate Beta-glucuronidasep-Hydroxyaniline mustard-glucuronide Epirubicin-glucuronidePenicillin-V-amidase Adriamycin-N phenoxyacetyl Penicillin-G-amidaseN-(4′-hydroxyphenyl acetyl) palytoxin Doxorubicin and melphalanBeta-lactamase Nitrogen mustard-cephalosporin p-phenylenediamine;doxorubicin derivatives; vinblastine derivative-cephalosporin,cephalosporin mustard; a taxol derivative Beta-glucosidaseCyanophenylmethyl-beta-D-gluco- pyranosiduronic acid Nitroreductase5-(Azaridin-1-yl-)-2,4-dinitrobenzamide Cytosine deaminase5-Fluorocytosine Carboxypeptidase A Methotrexate-alanine

(This table is adapted from Bagshawe (1995) Drug Dev. Res. 34, 220-230,from which full references for these various systems may be obtained;the taxol derivative is described in Rodrigues, M. L. et al (1995)Chemistry & Biology 2, 223).

Suitable enzymes for forming part of the enzymatic portion of theinvention include: exopeptidases, such as carboxypeptidases G, G1 and G2(for glutamylated mustard prodrugs), carboxypeptidases A and B (forMTX-based prodrugs) and aminopeptidases (for 2-α-aminocyl MTC prodrugs);endopeptidases, such as e.g. thrombolysin (for thrombin prodrugs);hydrolases, such as phosphatases (e.g. alkaline phosphatase) orsulphatases (e.g. aryl sulphatases) (for phosphylated or sulphatedprodrugs); amidases, such as penicillin amidases and arylacyl amidase;lactamases, such as β-lactamases; glycosidases, such as β-glucuronidase(for β-glucuronomide anthracyclines), α-galactosidase (for amygdalin)and β-galactosidase (for β-galactose anthracycline); deaminases, such ascytosine deaminase (for 5FC); kinases, such as urokinase and thymidinekinase (for gancyclovir); reductases, such as nitroreductase (for CB1954and analogues), azoreductase (for azobenzene mustards) and DT-diaphorase(for CB1954); oxidases, such as glucose oxidase (for glucose), xanthineoxidase (for xanthine) and lactoperoxidase; DL-racemases, catalyticantibodies and cyclodextrins.

The prodrug is relatively non-toxic compared to the cytotoxic drug.Typically, it has less than 10% of the toxicity, preferably less than 1%of the toxicity as measured in a suitable in vitro cytotoxicity test. Itis likely that the moiety which is able to convert a prodrug to acytotoxic drug will be active in isolation from the rest of the compoundbut it is necessary only for it to be active when (a) it is incombination with the rest of the compound and (b) the compound isattached to, adjacent to or internalised in target cells.

When each moiety of the compound is a polypeptide, the two portions maybe linked together by any of the conventional ways of cross-linkingpolypeptides, such as those generally described in O'Sullivan et al(1979) Anal. Biochem. 100, 100-108. Alternatively, the compound may beproduced as a fusion compound by recombinant DNA techniques whereby alength of DNA comprises respective regions encoding the two moieties ofthe compound of the invention either adjacent one another or separatedby a region encoding a linker peptide which does not destroy the desiredproperties of the compound. Conceivably, the two portions of thecompound may overlap wholly or partly.

The cytotoxic moiety may be a radiosensitizer. Radiosensitizers includefluoropyrimidines, thymidine analogues, hydroxyurea, gemcitabine,fludarabine, nicotinamide, halogenated pyrimidines, 3-aminobenzamide,3-aminobenzodiamide, etanixadole, pimonidazole and misonidazole (see,for example, McGinn et al (1996) J. Natl. Cancer Inst. 88, 1193-11203;Shewach & Lawrence (1996) Invest. New Drugs 14, 257-263; Horsman (1995)Acta Oncol. 34, 571-587; Shenoy & Singh (1992) Clin. Invest. 10,533-551; Mitchell et al (1989) Int. J. Radiat. Biol. 56, 827-836;Iliakis & Kurtzman (1989) Int. J. Radiat. Oncol. Biol. Phys. 16,1235-1241; Brown (1989) Int. J. Radiat. Oncol. Biol. Phys. 16, 987-993;Brown (1985) Cancer 55, 2222-2228).

Also, delivery of genes into cells can radiosensitise them, for exampledelivery of the p53 gene or cyclin D (Lang et al (1998) J. Neurosurg.89, 125-132; Coco Martin et al (1999) Cancer Res. 59, 1134-1140).

The further moiety may be one which becomes cytotoxic, or releases acytotoxic moiety, upon irradiation. For example, the boron-10 isotope,when appropriately irradiated, releases a particles which are cytotoxic(see for example, U.S. Pat. No. 4,348,376 to Goldenberg; Primus et al(1996) Bioconjug. Chem. 7, 532-535).

Similarly, the cytotoxic moiety may be one which is useful inphotodynamic therapy such as photofrin (see, for example, Dougherty etal (1998) J. Natl. Cancer Inst. 90, 889-905).

The cytotoxic moiety may be a nucleic acid molecule which is directly orindirectly cytotoxic. For example, the nucleic acid molecule may be anantisense oligonucleotide which, upon localisation at the target site isable to enter cells and lead to their death. The oligonucleotide,therefore, may be one which prevents expression of an essential gene, orone which leads to a change in gene expression which causes apoptosis.

Examples of suitable oligonucleotides include those directed at bcl-2(Ziegler et al (1997) J. Natl. Cancer Inst. 89, 1027-1036), and DNApolymerase α and topoisomerase IIα (Lee et al (1996) Anticancer Res. 16,1805-1811.

Peptide nucleic acids may be useful in place of conventional nucleicacids (see Knudsen & Nielsen (1997) Anticancer Drugs 8, 113-118).

In an embodiment of the compound of the invention, the antibody orantigen-binding fragment thereof and the cytotoxic moiety may bepolypeptides which are fused. Thus, the invention further provides apolynucleotide encoding such a compound.

In a further embodiment, the further moiety may be a readily detectablemoiety. It is envisaged that readily detectable moiety may comprise asuitable amount of any one of iodine-123, iodine-131, indium-111,fluorine-19, carbon-13, nitrogen-15, oxygen-17, technetium-99m,gadolinium, manganese or iron.

By a “readily detectable moiety” we include the meaning that the moietyis one which, when located at the target site following administrationof the compound of the invention into a patient, may be detected,typically non-invasively from outside the body and the site of thetarget located. Thus, the compounds of this embodiment of the inventionare useful in imaging and diagnosis.

Typically, the readily detectable moiety is or comprises a radioactiveatom which is useful in imaging. Suitable radioactive atoms includetechnetium-99m or iodine-123 for scintigraphic studies. Other readilydetectable moieties include, for example, spin labels for magneticresonance imaging (MRI) such as iodine-123 again, iodine-131,indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium,manganese or iron. Clearly, the compound of the invention must havesufficient of the appropriate atomic isotopes in order for the moleculeto be readily detectable.

The radio- or other labels may be incorporated in the compound of theinvention in known ways. For example, if the antibody is a polypeptideit may be biosynthesised or may be synthesised by chemical amino acidsynthesis using suitable amino acid precursors involving, for example,fluorine-19 in place of hydrogen. Labels such as ^(99m)Tc, ¹²³I, ¹⁸⁶Rh,¹⁸⁸Rh and ¹¹¹In can, for example, be attached via cysteine residues inthe antibody. Yttrium-90 can be attached via a lysine residue. TheIODOGEN method (Fraker er al (1978) Biochem. Biophys. Res. Comm. 80,49-57) can be used to incorporate iodine-123. Reference (“MonoclonalAntibodies in Immunoscintigraphy”, J-F Chatal, CRC Press, 1989)describes other methods in detail.

In a further aspect, the present invention provides a vector comprisingany polynucleotide of the invention.

Typical prokaryotic vector plasmids are: pUC18, pUC19, pBR322 and pBR329available from Biorad Laboratories (Richmond, Calif., USA); pTrc99A,pKK223-3, pKK233-3, pDR540 and pRIT5 available from Pharmacia(Piscataway, N.J., USA); pBS vectors, Phagescript vectors, Bluescriptvectors, pNH8A, pNH16A, pNH18A, pNH46A available from Stratagene CloningSystems (La Jolla, Calif. 92037, USA).

A typical mammalian cell vector plasmid is pSVL available from Pharmacia(Piscataway, N.J., USA). An example of an inducible mammalian expressionvector is pMSG, also available from Pharmacia (Piscataway, N.J., USA).

Useful yeast plasmid vectors are pRS403-406 and pRS413-416 and aregenerally available from Stratagene Cloning Systems (La Jolla, Calif.92037, USA). Plasmids pRS403, pRS404, pRS405 and pRS406 are YeastIntegrating plasmids (YIps) and incorporate the yeast selectable markersHIS3, TRP1, LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromereplasmids (YCps).

Methods well known to those skilled in the art can be used to constructexpression vectors containing the coding sequence and, for exampleappropriate transcriptional or translational controls.

Yet further, the invention provides a host cell comprising anypolynucleotide or vector of the invention.

Many expression systems are known, including systems employing: bacteria(e.g. E. coli and Bacillus subtilis) transformed with, for example,recombinant bacteriophage, plasmid or cosmid DNA expression vectors;yeasts (e.g. Saccharomyces cerevisiae) transformed with, for example,yeast expression vectors; insect cell systems transformed with, forexample, viral expression vectors (e.g. baculovirus); plant cell systemstransfected with, for example viral or bacterial expression vectors;animal cell systems transfected with, for example, adenovirus expressionvectors.

The vectors can include a prokaryotic replicon, such as the Col E1 ori,for propagation in a prokaryote, even if the vector is to be used forexpression in other, non-prokaryotic cell types. The vectors can alsoinclude an appropriate promoter such as a prokaryotic promoter capableof directing the expression (transcription and translation) of the genesin a bacterial host cell, such as E. coli, transformed therewith.

A promoter is an expression control element formed by a DNA sequencethat permits binding of RNA polymerase and transcription to occur.Promoter sequences compatible with exemplary bacterial hosts aretypically provided in plasmid vectors containing convenient restrictionsites for insertion of a DNA segment of the present invention.

The polynucleotide in a suitable host cell may be expressed to producethe antibody or compound of the invention. Thus, the polynucleotide maybe used in accordance with known techniques, appropriately modified inview of the teachings contained herein, to construct an expressionvector, which is then used to transform an appropriate host cell for theexpression and production of the antibody or compound of the invention.Such techniques include those disclosed in U.S. Pat. Nos. 4,440,859;4,530,901; 4,582,800; 4,677,063; 4,678,751; 4,704,362; 4,710,463;4,757,006; 4,766,075; and 4,810,648, all of which are incorporatedherein by reference.

The polynucleotide may be joined to a wide variety of other DNAsequences for introduction into an appropriate host. The companion DNAwill depend upon the nature of the host, the manner of the introductionof the DNA into the host, and whether episomal maintenance orintegration is desired.

Generally, the polynucleotide is inserted into an expression vector,such as a plasmid, in proper orientation and correct reading frame forexpression. If necessary, the DNA may be linked to the appropriatetranscriptional and translational regulatory control nucleotidesequences recognised by the desired host, although such controls aregenerally available in the expression vector. Thus, the DNA insert maybe operatively linked to an appropriate promoter. Bacterial promotersinclude the E. coli lacI and lacZ promoters, the T3 and T7 promoters,the gpt promoter, the phage λ PR and PL promoters, the phoA promoter andthe trp promoter. Eukaryotic promoters include the CMV immediate earlypromoter, the HSV thymidine kinase promoter, the early and late SV40promoters and the promoters of retroviral LTRs. Other suitable promoterswill be known to the skilled artisan. The expression constructs willdesirably also contain sites for transcription initiation andtermination, and in the transcribed region, a ribosome binding site fortranslation. (Hastings et al, International Patent No. WO 98/16643,published 23 Apr. 1998)

The vector is then introduced into the host through standard techniques.Generally, not all of the hosts will be transformed by the vector and itwill therefore be necessary to select for transformed host cells. Oneselection technique involves incorporating into the expression vector aDNA sequence marker, with any necessary control elements, that codes fora selectable trait in the transformed cell. These markers includedihydrofolate reductase, G418 or neomycin resistance for eukaryotic cellculture, and tetracyclin, kanamycin or ampicillin resistance genes forculturing in E. coli and other bacteria. Alternatively, the gene forsuch selectable trait can be on another vector, which is used toco-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the polypeptide, which canthen be recovered.

The antibody or compound can be recovered and purified from recombinantcell cultures by well-known methods including ammonium sulphate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Most preferably, high performance liquidchromatography (“HPLC”) is employed for purification.

Yet further, the invention provides a stable host cell line producing anantibody or antigen-binding fragment thereof according to any precedingaspect or a compound of the invention resulting from incorporation inthe cell line an exogenous polynucleotide or vector of the invention.

The host cell can be either prokaryotic or eukaryotic. Bacterial cellsare preferred prokaryotic host cells and typically are a strain ofEscherichia coli such as, for example, the E. coli strains DH5 availablefrom Bethesda Research Laboratories Inc., Bethesda, Md., USA, and RR1available from the American Type Culture Collection (ATCC) of Rockville,Md., USA (No ATCC 31343). Preferred eukaryotic host cells include yeastand mammalian cells, preferably vertebrate cells such as those from amouse, rat, monkey or human fibroblastic cell line. Yeast host cellsinclude YPH499, YPH500 and YPH501 which are generally available fromStratagene Cloning Systems, La Jolla, Calif. 92037, USA. Preferredmammalian host cells include Chinese hamster ovary (CHO) cells availablefrom the ATCC as CCL61, NIH Swiss mouse embryo cells NIH/3T3 availablefrom the ATCC as CRL 1658, and monkey kidney-derived COS-1 cellsavailable from the ATCC as CRL 1650.

In addition to the transformed host cells themselves, the presentinvention also contemplates a culture of those cells, preferably amonoclonal (clonally homogeneous) culture, or a culture derived from amonoclonal culture, in a nutrient medium.

In a further aspect, the present invention provides a pharmaceuticalcomposition/formulation comprising an antibody or antigen-bindingfragment thereof according to any aspect of the invention, or apolynucleotide according to the invention, or a compound according tothe invention, in admixture with a pharmaceutically acceptableexcipient, adjuvant, diluent or carrier.

By “pharmaceutically acceptable” is included that the formulation issterile and pyrogen free. Suitable pharmaceutical carriers are wellknown in the art of pharmacy. The carrier(s) must be “acceptable” in thesense of being compatible with the compound of the invention and notdeleterious to the recipients thereof. Typically, the carriers will bewater or saline which will be sterile and pyrogen free; however, otheracceptable carriers may be used.

In an embodiment, the pharmaceutical composition/formulation of theinvention may further comprise a further active ingredient, i.e. atherapeutically active agent other than the antibody or antigen-bindingfragment thereof of the invention. It is envisaged that one or moreadditional active agents may increase the efficacy of the pharmaceuticalcomposition/formulation against the targeted disease as appropriate. Inan embodiment, the further active ingredient may be a therapeutic agentselected from an agent involved in reducing tissue scarring, reducingneurofibrillary tangles, and/or reducing resistance to chemotherapy.

In a preferred embodiment, pharmaceutical composition/formulation may beformulated for intravenous, intramuscular, or subcutaneous delivery to apatient.

It is preferred that the pharmaceutical composition/formulationcomprises an amount of the antibody or antigen-binding fragment of theinvention effective to treat the various conditions described above andfurther below.

A further aspect of the invention provides a kit of parts comprising anantibody or antigen-binding fragment thereof according to any aspect ofthe invention, or a polynucleotide according of the invention, or acompound of the invention; and one or more further agents. It isenvisaged that the further agents may be any one of the further activeingredients described above, or any other suitable agent.

In a yet further aspect, the invention provides a therapeuticallyeffective amount of an antibody or antigen-binding fragment thereofaccording to any aspect of the invention, or a polynucleotide of theinvention, or a compound, pharmaceutical composition/formulation, or kitof parts of the invention, for use in medicine.

TG2 clearly is a multifunction enzyme and has been linked to a range ofcellular functions including nuclear stabilisation and transport [28,29], endocytosis [30, 31], GTPase signalling [32-34], Apoptosis [35,36], cell adhesion [37-39], cytoskeletal integrity [28, 29] and ECMstabilisation [9]. A small molecule inhibitor may impede on all of thesefunctions as in general they have free access to the extracellular spaceand cell interior. An antibody cannot enter the cell and as such theintracellular roles of TG2 would not be affected by a TG2 specificantibody administered in vivo.

Importantly most of the pathological roles of TG2 appear to beextracellular such as its role in tissue scarring and fibrosis, celiacdisease and cancer. Thus using an antibody which selectively binds TG2in medicine would bring an additional degree of selectivity preventingundesired intracellular effects that could translate into undesired sideeffects of therapy.

Therefore the antibodies and antigen-binding fragments thereof of theinvention would offer clinical advantages over previously availabledrugs, for example in blocking TG2 in fibrotic and scarring diseaseswhere TG2 crosslinks ECM proteins, in celiac disease where gliadin isdeamidated in the extracellular space and in chemo-resistance in cancerwhere cell adhesion appears to be the protective factor. Further, thesmall antibody fragments of the invention, for example the Fab fragmentscould cross the blood brain barrier and inhibit TG2 in the brain andpotentially offer effective therapies for neurological pathologies withTG2 involvement.

Thus, a further aspect of the invention provides an antibody orantigen-binding fragment thereof according to any aspect of theinvention, or a polynucleotide of the invention, or a compound,pharmaceutical composition/formulation, or kit of parts of theinvention, for use in reducing or inhibiting TG2 enzyme activity in anindividual in need thereof.

The invention further provides for the use of an antibody orantigen-binding fragment thereof according to any aspect of theinvention, or a polynucleotide of the invention, or a compound,pharmaceutical composition/formulation, or kit of parts of theinvention, in the manufacture of a medicament for reducing or inhibitingTG2 enzyme activity in an individual in need thereof.

The invention also provides a method of reducing or inhibiting TG2enzyme activity in an individual in need thereof, the method comprisingthe step of administering an antibody or antigen-binding fragmentthereof, or a variant, fusion or derivative thereof according to anyaspect of the invention, or a polynucleotide of the invention, or acompound, pharmaceutical composition/formulation, or kit of parts of theinvention, to the individual.

A further aspect of the invention provides a therapeutically effectiveamount of an antibody or antigen-binding fragment thereof according toany aspect of the invention, or a polynucleotide of the invention, or acompound, pharmaceutical composition/formulation, or kit of parts of theinvention, for use in the treatment and/or diagnosis of Celiac disease,abnormal wound healing, scarring, scleroderma, keloids and hypertrophicscars, ocular scarring, inflammatory bowel disease, maculardegeneration, Grave's opthalmopathy, drug-induced ergotism, psoriasis,fibrosis-related diseases (e.g. liver fibrosis, pulmonary fibrosis suchas interstitial lung disease and fibrotic lung disease, cardiacfibrosis, skin fibrosis, myelofibrosis, kidney fibrosis such asglomerulosclerosis and tubulointerstitial fibrosis), atherosclerosis,restenosis, inflammatory diseases, autoimmune diseases,neurodegenerative/neurological diseases (e.g. Huntington's Disease,Alzheimer's disease, Parkinson's disease, polyglutamine disease,spinobulbar muscular atrophy, dentatorubral-pallidoluysian atrophy,spinocerebellar ataxias 1, 2, 3, 6, 7 and 12, rubropallidal atrophy,spinocerebellar palsy), and/or cancer (e.g. glioblastomas such asglioblastoma in Li-Fraumeni syndrome and sporadic glioblastoma,malignant melanomas, pancreatic ductal adenocarcinomas, myeloidleukemia, acute myelogenous leukemia, myelodysplastic syndrome,myeloproliferative syndrome, gynaecological cancer, Kaposi's sarcoma,Hansen's disease, collagenous colitis).

The invention also provides for the use of a therapeutically effectiveamount of an antibody or antigen-binding fragment thereof according toany aspect of the invention, or a polynucleotide of the invention, or acompound, pharmaceutical composition/formulation, or kit of parts of theinvention, in the manufacture of a medicament for treating and/ordiagnosing Celiac disease, abnormal wound healing, scarring,scleroderma, keloids and hypertrophic scars, ocular scarring,inflammatory bowel disease, macular degeneration, Grave's opthalmopathy,drug-induced ergotism, psoriasis, fibrosis-related diseases (e.g. liverfibrosis, pulmonary fibrosis such as interstitial lung disease andfibrotic lung disease, cardiac fibrosis, skin fibrosis, myelofibrosis,kidney fibrosis such as glomerulosclerosis and tubulointerstitialfibrosis), atherosclerosis, restenosis, inflammatory diseases,autoimmune diseases, neurodegenerative/neurological diseases (e.g.Huntington's Disease, Alzheimer's disease, Parkinson's disease,polyglutamine disease, spinobulbar muscular atrophy,dentatorubral-pallidoluysian atrophy, spinocerebellar ataxias 1, 2, 3,6, 7 and 12, rubropallidal atrophy, spinocerebellar palsy), and/orcancer (e.g. glioblastomas such as glioblastoma in Li-Fraumeni syndromeand sporadic glioblastoma, malignant melanomas, pancreatic ductaladenocarcinomas, myeloid leukemia, acute myelogenous leukemia,myelodysplastic syndrome, myeloproliferative syndrome, gynaecologicalcancer, Kaposi's sarcoma, Hansen's disease, collagenous colitis).

The invention also provides a method of treating and/or diagnosingCeliac disease, abnormal wound healing, scarring, scleroderma, keloidsand hypertrophic scars, ocular scarring, inflammatory bowel disease,macular degeneration, Grave's opthalmopathy, drug-induced ergotism,psoriasis, fibrosis-related diseases (e.g. liver fibrosis, pulmonaryfibrosis such as interstitial lung disease and fibrotic lung disease,cardiac fibrosis, skin fibrosis, myelofibrosis, kidney fibrosis such asglomerulosclerosis and tubulointerstitial fibrosis), atherosclerosis,restenosis, inflammatory diseases, autoimmune diseases,neurodegenerative/neurological diseases (e.g. Huntington's Disease,Alzheimer's disease, Parkinson's disease, polyglutamine disease,spinobulbar muscular atrophy, dentatorubral-pallidoluysian atrophy,spinocerebellar ataxias 1, 2, 3, 6, 7 and 12, rubropallidal atrophy,spinocerebellar palsy), and/or cancer (e.g. glioblastomas such asglioblastoma in Li-Fraumeni syndrome and sporadic glioblastoma,malignant melanomas, pancreatic ductal adenocarcinomas, myeloidleukemia, acute myelogenous leukemia, myelodysplastic syndrome,myeloproliferative syndrome, gynaecological cancer, Kaposi's sarcoma,Hansen's disease, collagenous colitis) in a patient, the methodcomprising administering a therapeutically effective amount of anantibody or antigen-binding fragment thereof according to any aspect ofthe invention, or a polynucleotide of the invention, or a compound,pharmaceutical composition/formulation, or kit of parts of theinvention, to the patient.

By “treatment” we include both therapeutic and prophylactic treatment ofa subject/patient. The term “prophylactic” is used to encompass the useof an antibody, medicament, compound, composition, or kit describedherein which either prevents or reduces the likelihood of the occurrenceor development of a condition or disorder (such as a fibrosis-relateddisorder) in an individual.

It is preferred that the patient is a human but the patient may be anyother mammal that may benefit from the treatment. For example, thepatient may be a mouse, a rat, a hamster, a rabbit, a cat, a dog, agoat, a sheep, a monkey or an ape.

A “therapeutically effective amount”, or “effective amount”, or“therapeutically effective”, as used herein, refers to that amount whichprovides a therapeutic effect for a given condition and administrationregimen. This is a predetermined quantity of active material calculatedto produce a desired therapeutic effect in association with the requiredadditive and diluent, i.e. a carrier or administration vehicle. Further,it is intended to mean an amount sufficient to reduce or prevent aclinically significant deficit in the activity, function and response ofthe host. Alternatively, a therapeutically effective amount issufficient to cause an improvement in a clinically significant conditionin a host, for example a mammal.

The agents (i.e. antibody, antigen-binding fragment, variant, fusion orderivative thereof), medicaments, compounds, pharmaceuticalcompositions/formulations and kits of the invention may be deliveredusing an injectable sustained-release drug delivery system. These aredesigned specifically to reduce the frequency of injections. An exampleof such a system is Nutropin Depot which encapsulates recombinant humangrowth hormone (rhGH) in biodegradable microspheres that, once injected,release rhGH slowly over a sustained period. Preferably, delivery isperformed intra-muscularly (i.m.) and/or sub-cutaneously (s.c.) and/orintravenously (i.v.).

The agents, medicaments, compounds, pharmaceuticalcompositions/formulations and kits of the invention can be administeredby a surgically implanted device that releases the drug directly to therequired site. For example, Vitrasert releases ganciclovir directly intothe eye to treat CMV retinitis. The direct application of this toxicagent to the site of disease achieves effective therapy without thedrug's significant systemic side-effects.

Preferably, the medicaments and/or pharmaceuticalcompositions/formulations of the present invention is a unit dosagecontaining a daily dose or unit, daily sub-dose or an appropriatefraction thereof, of the active ingredient(s).

The agents, medicaments, compounds, pharmaceutical compositions and kitsof the invention will normally be administered by any parenteral route,in the form of a pharmaceutical composition comprising the activeingredient(s), optionally in the form of a non-toxic organic, orinorganic, acid, or base, addition salt, in a pharmaceuticallyacceptable dosage form. Depending upon the disorder and patient to betreated, as well as the route of administration, the compositions may beadministered at varying doses.

In human therapy, the agents, medicaments, compounds, pharmaceuticalcompositions/formulations, and kits of the invention can be administeredalone but will generally be administered in admixture with a suitablepharmaceutical excipient, diluent or carrier selected with regard to theintended route of administration and standard pharmaceutical practice.

The agents, medicaments, compounds, pharmaceuticalcompositions/formulations and kits of the invention can be administeredparenterally, for example, intravenously, intra-arterially,intraperitoneally, intra-thecally, intra-muscularly or subcutaneously,or they may be administered by infusion techniques. They are best usedin the form of a sterile aqueous solution which may contain othersubstances, for example, enough salts or glucose to make the solutionisotonic with blood. The aqueous solutions should be suitably buffered(preferably to a pH of from 3 to 9), if necessary. The preparation ofsuitable parenteral formulations under sterile conditions is readilyaccomplished by standard pharmaceutical techniques well-known to thoseskilled in the art.

Medicaments and pharmaceutical compositions suitable for parenteraladministration include aqueous and non-aqueous sterile injectionsolutions which may contain anti-oxidants, buffers, bacteriostats andsolutes which render the formulation isotonic with the blood of theintended recipient; and aqueous and non-aqueous sterile suspensionswhich may include suspending agents and thickening agents. Themedicaments and pharmaceutical compositions may be presented inunit-dose or multi-dose containers, for example sealed ampoules andvials, and may be stored in a freeze-dried (lyophilised) conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets of the kind previously described.

For parenteral administration to human patients, the daily dosage levelof the agents, medicaments and pharmaceutical compositions of theinvention will usually be from 1 μg to 10 mg per adult per dayadministered in single or divided doses. The physician in any event willdetermine the actual dosage which will be most suitable for anyindividual patient and it will vary with the age, weight and response ofthe particular patient. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited and such are within the scope of thisinvention.

Typically, the medicaments, pharmaceutical compositions/formulations andkits of the invention will contain the agent of the invention at aconcentration of between approximately 2 mg/ml and 150 mg/ml or betweenapproximately 2 mg/ml and 200 mg/ml. In a preferred embodiment, themedicaments, pharmaceutical compositions/formulations and kits of theinvention will contain the agent of the invention at a concentration of10 mg/ml.

Generally, in humans, parenteral administration of the agents,medicaments, compounds, pharmaceutical compositions/formulations andkits of the invention is the preferred route, being the most convenient.

For veterinary use, the agents, medicaments, compounds, pharmaceuticalcompositions/formulations, and kits of the invention are administered asa suitably acceptable formulation in accordance with normal veterinarypractice and the veterinary surgeon will determine the dosing regimenand route of administration which will be most appropriate for aparticular animal.

The present invention also includes pharmaceuticalcompositions/formulations comprising pharmaceutically acceptable acid orbase addition salts of the polypeptide binding moieties of the presentinvention. The acids which are used to prepare the pharmaceuticallyacceptable acid addition salts of the aforementioned base compoundsuseful in this invention are those which form non-toxic acid additionsalts, i.e. salts containing pharmacologically acceptable anions, suchas the hydrochloride, hydrobromide, hydroiodide, nitrate, sulphate,bisulphate, phosphate, acid phosphate, acetate, lactate, citrate, acidcitrate, tartrate, bitartrate, succinate, maleate, fumarate, gluconate,saccharate, benzoate, methanesulphonate, ethanesulphonate,benzenesulphonate, p-toluenesulphonate and pamoate [i.e.1,1′-methylene-bis-(2-hydroxy-3 naphthoate)] salts, among others.

Pharmaceutically acceptable base addition salts may also be used toproduce pharmaceutically acceptable salt forms of the agents (i.e.antibody or antigen-binding fragment thereof) according to the presentinvention.

The chemical bases that may be used as reagents to preparepharmaceutically acceptable base salts of the present agents that areacidic in nature are those that form non-toxic base salts with suchcompounds. Such non-toxic base salts include, but are not limited tothose derived from such pharmacologically acceptable cations such asalkali metal cations (e.g. potassium and sodium) and alkaline earthmetal cations (e.g. calcium and magnesium), ammonium or water-solubleamine addition salts such as N-methylglucamine-(meglumine), and thelower alkanolammonium and other base salts of pharmaceuticallyacceptable organic amines, among others.

The agents and/or polypeptide binding moieties of the invention may belyophilised for storage and reconstituted in a suitable carrier prior touse. Any suitable lyophilisation method (e.g. spray drying, cake drying)and/or reconstitution techniques can be employed. It will be appreciatedby those skilled in the art that lyophilisation and reconstitution canlead to varying degrees of antibody activity loss (e.g. withconventional immunoglobulins, IgM antibodies tend to have greateractivity loss than IgG antibodies) and that use levels may have to beadjusted upward to compensate. In one embodiment, the lyophilised(freeze dried) polypeptide binding moiety loses no more than about 20%,or no more than about 25%, or no more than about 30%, or no more thanabout 35%, or no more than about 40%, or no more than about 45%, or nomore than about 50% of its activity (prior to lyophilisation) whenre-hydrated.

Preferably, the invention provides an antibody, compound, pharmaceuticalcomposition/formulation, kit, use or method wherein the effective amountof the antibody or antigen-binding fragment thereof is between about0.0001 mg/kg to 50 mg/kg of the antibody or antigen-binding fragmentthereof.

As is appreciated by those skilled in the art, the precise amount of acompound may vary depending on its specific activity. Suitable dosageamounts may contain a predetermined quantity of active compositioncalculated to produce the desired therapeutic effect in association withthe required diluent. In the methods and use for manufacture ofcompositions of the invention, a therapeutically effective amount of theactive component is provided. A therapeutically effective amount can bedetermined by the ordinary skilled medical or veterinary worker based onpatient characteristics, such as age, weight, sex, condition,complications, other diseases, etc., as is well known in the art.

A further aspect of the invention provides an in vitro method ofreducing or inhibiting TG2 enzyme activity, the method comprisingadministering an antibody or antigen-binding fragment thereof accordingto any aspect of the invention, or a polynucleotide according to theinvention, or a compound or kit of the invention, to a sample comprisingTG2.

The “sample” may be any sample obtained from an appropriate source, forexample a mammalian source. For example, the sample may be a tissue orcell sample comprising TG2. Exemplary tissues include tissue obtainedfrom a patient's brain, gastro-intestinal tract, lung, pancreas, liver,skin, kidney, eye, heart, blood vessels, lymph nodes, spine, andskeletal or smooth muscle.

The invention also provides a method of reducing or inhibiting TG2enzyme activity in an individual in need thereof, the method comprisingadministering an effective amount of a polynucleotide encoding anantibody or antigen-binding fragment thereof according to any aspect ofthe invention, to the individual.

A further aspect provides the use of a polynucleotide encoding anantibody or antigen-binding fragment thereof according to any aspect ofthe invention, in the manufacture of a medicament for reducing orinhibiting TG2 enzyme activity in an individual in need thereof.

The invention also provides an in vitro method of reducing or inhibitingTG2 enzyme activity, the method comprising administering apolynucleotide encoding an antibody or antigen-binding fragment thereofaccording to any aspect of the invention, to a sample comprising TG2,for example a tissue or cell sample comprising TG2.

In a yet further aspect, the invention provides a method of producing anantibody or antigen-binding fragment according to the second aspect ofthe invention, or a compound of the invention comprising an antibody orantigen-binding fragment according to the second aspect of theinvention, the method comprising expressing a polynucleotide of theinvention, or culturing a stable host cell line of the invention.

In a further aspect, the invention provides a method of selecting anantibody or antigen-binding fragment thereof that selectively binds atransglutaminase protein, the method comprising the step of selecting anantibody or antigen-binding fragment thereof that selectively binds apolypeptide comprising a transglutaminase core region/catalytic domainbut not comprising a transglutaminase barrel or sandwich domain.

In a further aspect, the invention provides a method of selecting anantibody or antigen-binding fragment thereof according to the first orsecond aspects of the invention, or a compound of the inventioncomprising an antibody or antigen-binding fragment according to thefirst or second aspect of the invention, the method comprising the stepof selecting an antibody or antigen-binding fragment thereof thatselectively binds a polypeptide sequence consisting of the polypeptidesequence of amino acids 143 to 473 of human TG2 or a fragment thereof.

In an embodiment, the method may be carried out using antibody phagedisplay. It is preferred that the antibody or antigen-binding fragmentthereof is an inhibitory antibody that inhibits catalytic activity ofthe transglutaminase protein.

In a further aspect, the invention provides a method of producing anantibody or antigen-binding fragment thereof that selectively binds atransglutaminase protein, the method comprising administering to anon-human animal a compound comprising:

-   -   i) a polypeptide comprising a transglutaminase core        region/catalytic domain but not comprising a transglutaminase        barrel or sandwich domain, or a fragment thereof; and,        optionally,    -   ii) an adjuvant.

It is envisaged that the polypeptide comprising a transglutaminase coreregion/catalytic domain but not comprising a transglutaminase barrel orsandwich domain will comprise the catalytic triad described above, andoptionally, also the GTP binding site of the transglutaminase protein.

In an embodiment, the method may further comprise the step of selectingan antibody or antigen-binding fragment thereof on the basis of itsselective binding to a transglutaminase protein.

In a further aspect, the invention provides a method of selecting anantibody or antigen-binding fragment thereof that selectively binds atransglutaminase protein, the method comprising the step of selecting anantibody or antigen-binding fragment thereof that selectively binds apolypeptide sequence consisting of the polypeptide sequence of aminoacids 143 to 473 of human TG2 or a fragment thereof.

In a yet further aspect, the invention provides a method of producing anantibody or antigen-binding fragment according to any aspect of theinvention comprising administering to a non-human animal a compoundcomprising:

-   -   i) a polypeptide sequence consisting of the polypeptide sequence        of amino acids 143 to 473 of human TG2 or a fragment thereof;        and optionally,    -   ii) an adjuvant.

In an embodiment, the method may further comprise the step of selectingan antibody or antigen-binding fragment thereof on the basis of itsselective binding to TG2, for example human TG2.

In a further aspect, the invention provides an antibody orantigen-binding fragment thereof obtainable by any of the precedingmethods of producing or selecting an antibody or antigen-bindingfragment thereof.

By “adjuvant” we include any a pharmacological or immunological agentthat enhances the recipient's immune response to the polypeptide of theinvention. Immunologic adjuvants are added to vaccines to stimulate theimmune system's response to the target antigen, but do not in themselvesconfer immunity. Examples of adjuvants include oil emulsions, inorganiccompounds such as aluminium salts, for example aluminum hydroxide oraluminium phosphate, organic compounds such as Squalene, virosomes, orany other suitable compound or compounds as would be understood by aperson of skill in the art.

In a further aspect, the invention provides an isolated polypeptideconsisting of:

-   -   i) the polypeptide sequence of amino acids 143 to 473 of human        TG2;    -   ii) the polypeptide sequence of amino acids 304 to 326 of human        TG2;    -   iii) the polypeptide sequence of amino acids 351 to 365 of human        TG2;    -   iv) the polypeptide sequence of amino acids 450 to 467 of human        TG2; or    -   a fragment, derivative or polypeptide mimic thereof.

The invention also provides an isolated polynucleotide encoding thepolypeptide of the immediately preceding aspect.

The invention provides an antibody or antigen-binding fragment thereoffor use in treating a condition associated with TG2 activitysubstantially as described herein with reference to the description andfigures.

The invention also provides the use of an antibody or antigen-bindingfragment thereof substantially as described herein with reference to thedescription and figures.

As used herein, the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “an antibody” includes a plurality of suchantibodies and reference to “the dosage” includes reference to one ormore dosages and equivalents thereof known to those skilled in the art,and so forth.

All documents referred to herein are hereby incorporated by reference.

The invention is now described in more detail by reference to thefollowing, non-limiting, Figures and Examples.

FIG. 1: Generation of a human TG2 recombinant protein

A: The TG2 catalytic core cDNA was generated by PCR from thepClineo-hTG2 vector and inserted into the pET 21a plasmid. Followingamplification in E. Coli this was digested with Nhe I and Hind III torelease the TG2 core cDNA and run on a 1% Agarose gel (lane 3). Bandswere sized by reference to a 100 bp ladder (lane 1) and λ DNA molecularweight marker (Lane 2).

B: The pET21a TG2 core vector was used to transform E. coli strainBL21-CodonPlus(DE3)-RIPL. Expression was induced using IPTG for 4 hours.TG2 core protein formed insoluble bodies that were recovered fromlysates by centrifugation. These were re solubilised, and the 37 kDa Histagged TG2 core purified on a nickel column. 10 ng was separated bySDS-PAGE, western blotted and probed with CUB7402 anti TG2 antibody(lane 2) with reference to a precision plus molecular weight marker(lane 1).

FIG. 2: Immunological Response in mice to rhTG2 core protein

A: Test bleeds were taken from 4 catalytic core immunised mice at day 45after the first immunisation and 10 days after the second boost. Serumwas serially diluted and reactivity checked by ELISA against immobilisedTG2 core protein.

B Reactivity was further checked by screened against human rh TG2 and rhTG2 catalytic core domain. 20, 40, 80 ng of protein was fractionated bySDS PAGE and western blotted onto a PVDF membrane. This was immunoprobedwith a 1:1000 dilution of serum. Antibody binding was revealed usinganti-mouse γ-chain specific HRP. For size reference to a precision plusmolecular weight marker was used.

FIG. 3: Hybridoma Reactivity Against TG family Members

A: ELISA were carried out using plates coated with recombinant TGs (100ng/well) to determine TG type specificity in 109 hybridoma supernatantsthat showed good reactivity to TG2. Antibody binding was revealed usinganti-mouse γ-chain specific HRP. A random selection of those screened isshown including EF4, CG9 & FD8 that showed cross reactivity.

B: Nine selected hybridomas were double cloned. IgG was purified andtested for reactivity at 0.1 ug/ml against recombinant human TG1, TG2,TG3, TG7 and Factor XIIIa using ELISA with plates coated with 100 ng ofeach TG. Data represents mean OD value from 3 separate ELISA±SEM.

Factor XIIIa is denoted on graphs as TG13.

FIG. 4: Identification of Hybridoma with inhibitory activity against TG2

Conditioned media from 32 hybridoma wells with specificity to TG2 werescreened for their effects on 100 ng of rhTG2 activity using the ³Hputrescine incorporation assay. The chemical pan TG2 inhibitor1,3-Dimethyl-2-[(2-oxo-propyl)thio]imidazolium chloride was used as apositive control for inhibition. RPMI (unconditioned medium) was used anegative control. 500 ng of a TG2 inhibitory antibody piloted by Quarkbiotechnology was included for comparison. Data represents mean CPMincorporated in 30 mins from at least three experiments done induplicate±SEM. Bars shown in grey show significant TG2 inhibition(p<0.05).

FIG. 5: Mapping of Inhibitory Antibody Epitopes.

Each inhibitory monoclonal antibody was bound to an ELISA plate andpanned against a human TG2 phage library. Phage binding to the antibodywere rescued, amplified and subjected to 4 further rounds of panning.TG2 library fragments in the phage were then sequenced and overlappingsequences used to determine the epitope for each antibody. Commonsequences between antibodies were then used to determine a consensussequence for a particular inhibitory epitope and antibodies groupedaccordingly. 3 inhibitory epitopes were identified.

FIG. 6: Structural location of Inhibitory Epitopes with the TG2catalytic Core

The TG2 amino acid sequence was entered into Pymol and a 3D graphicalrepresentation of the structure generated in its open, Ca²⁺ activatedstate with putative calcium binding sites (turquoise) and the catalytictriad (grey) shown for reference (left panel). The consensus inhibitoryepitopes were then added in blue (Antibody group 1—AB1 site), red(antibody group 2—DF4 site) and yellow (antibody group 3, DD9 site).

FIG. 7: VL Sequence of Inhibitory antibodies

RNA from each inhibitory hybridoma was extracted, reverse transcribedand amplified by PCR using a degenerate FR1 primers, MH1 and MH2 primersand 3 constant region primers to amplify VH genes. The resulting VH andVK sequences are shown for AB1.

FIG. 8. Efficacy of AB1 to inhibit TG2 activity in a cell homogenate

A: Hep2G cells were lysed and 45 ug of protein mixed with 750 ng of IgGfrom AB1, DH2, DD9, BB7, DC1 and EH6 for 20 minutes. This wassubsequently assayed using the ³H Putrescine incorporation TG activityassay with sampling over 1 hour. The rate of reaction was calculated andexpressed as a percentage of the same lysate incubated with a randomantibody (MAB002). Data represents the mean percentage inhibition±SEMfrom 2 separate experiments done in duplicate. * p<0.05

B: HepG2 cells were exposed to increasing glucose concentrations for 96hours to up regulate TG2 expression. Cells were harvested, lysed and 25ug of lysate fractionated by SDS-PAGE, western blotted and thenimmunoprobed with a 1 ng/ml solution of AB1 IgG using a chemiluminesantend point.

FIG. 9. (Table 1): Comparative IC50 values for TG2 inhibitory antibodies

To determine an IC50 value for each antibody against human, rat andmouse the ³H Putrescine assay was used. 100 ng of human TG2 or 25 ng ofmouse and rat TG2 was used to generate a reaction where approximately3000 cpm of Putrescine were incorporated per hour in 10 ul of thereaction mixture. Serial dilutions of each antibody were then appliedstarting from adding 500 ng (5 ug/ml final concentration) to thereaction mixture and incubated with the TG2 for 20 minutes prior toactivating the reaction. IC50 values were calculated by determining theconcentration at which the enzymatic rate of reaction was reduced by 50%using an appropriate curve fit in graphpad prism. Values are expressedas the amount of IgG in mg/ml in the reaction that would inhibit 1 ng ofTG2.

FIG. 10. Extracellular TG activity in HK2 cells in response to TG2inhibition.

HK2 cells were plated onto fibronectin and incubated for 2 hours in thepresence of 0.1M biotin cadaverine with either 4 ng/μl of human anti-TG2antibody (AB1) (part A), 4 ng/μl of human anti-TG2 antibody (DC1) (partB) or 400 μM of the site-specific pan TG inhibitor1,3-Dimethyl-2-[(2-oxo-propyl)thio]imidazolium chloride. ExtracellularTG activity was measured by the incorporation of biotin cadaverine intofibronectin with incorporation revealed using extravadin-HRP and a TMBsubstrate. Changes in optical density were measured at 450 nm in a 96well plate reader. Data represents mean OD at 450 nm corrected to 1 mgof cell protein. n=6 wells per experimental group.

FIG. 11. Comparison of TG2 inhibition by antibody AB1 to a fab fragmentof Quark's TG2 inhibitory antibody using a ³H putrescine incorporationassay

100 ng of hTG2 was assayed for TG2 activity based on the incorporationof ³H Putrescine into dimethylcasein over a 60 minute period with theaddition of either 1 μg of a fab fragment of an antibody described byQuark in WO2006/100679 and synthesised at Sheffield University or 500 ngof AB1. Data represents mean TG activity as incorporation of ³Hputrescine (CPM)±SEM from 3 independent experiments done in duplicate.

FIG. 12. Percentage comparison of TG2 inhibition by antibody AB1 with afab fragment of Quark's TG2 inhibitory antibody using a ³H putrescineincorporation assay

Data from FIG. 11 is alternatively expressed as a percentage of TGactivity at each time point to display the relative comparativeknockdown of TG2 activity by application of AB1 and the Quark antibodyfab fragment.

FIG. 13. Comparison of TG2 inhibition by antibody AB1 to a recombinantrat IgG of Quark's TG2 inhibitory antibody using a ³H putrescineincorporation assay

100 ng of hTG2 was assayed for TG2 activity based on the incorporationof ³H Putrescine into dimethylcasein over a 60 minute period with theaddition of either 500 ng of a recombinant rat version of a TG2inhibitory antibody described by Quark in WO2006/100679 and synthesisedat Medical Research Council Technology or 500 ng of AB1. Data representsmean TG activity as incorporation of ³H putrescine (CPM)±SEM from 3independent experiments done in duplicate.

FIG. 14. Percentage comparison of TG2 inhibition by antibody AB1 to arecombinant rat IgG of Quark's TG2 inhibitory antibody using a ³Hputrescine incorporation assay

Data from FIG. 13 is alternatively expressed as a percentage of TGactivity at each time point to display the relative comparativeknockdown of TG2 activity by application of AB1 and the Quarkrecombinant rat IgG.

FIG. 15. Effect of AB1 on ECM levels in HK2 cells

Mature collagen levels in HK-2 cells were measured by the incorporationof ³H proline into the ECM over a 76 hour period either with or withoutthe addition of TG2 inhibitory antibody AB1. Data represents theincorporation of ³H proline per mg of cellular protein expressed as apercentage of the mean level in untreated cells±SEM. n=2.

FIG. 16. Binding ELISA of humanised versions of antibodies.

Supernatants from HEK293F cells co-transfected with differentcombinations of humanised light chains and heavy chain vectors wereassayed in an anti-human IgG ELISA to determine concentration and in ananti huTG2 ELISA. Each supernatant was assayed in triplicate and IC₅₀'sdetermined. The most potent combination was selected for further studiesand as the candidate humanised antibody.

FIG. 17. Testing MRC Quark CTD190 on human Tg2 by ELISA.

96 well plates were plated with hTG2 (1 μg/ml) in carbonate bufferovernight and ELISA detection performed using 100 ng/ml primaryantibody. Detection was performed using anti-mouse IgG (SIGMA 3673) forCUB and anti-rat IgG (SIGMA A5795) for the Quark (both 1:5000). TheQuark antibody made by MRC T reacts with human TG2.

FIG. 18: RNA from the AB1 hybridoma was extracted, reverse transcribedand amplified by PCR using the degenerate signal sequence primer MHV4with heavy chain constant region primer MHCG1, or using the degeneratesignal sequence primer MKV4 with a kappa light chain constant regionprimer MKC. The resulting VH and VK sequences are shown.

FIG. 19: RNA from the BB7 hybridoma was extracted, reverse transcribedand amplified by PCR using the degenerate signal sequence primer MHV4with heavy chain constant region primer MHCG1, or using the degeneratesignal sequence primer MKV4 with a kappa light chain constant regionprimer MKC. The resulting VH and VK sequences are shown.

FIG. 20: RNA from the DC1 hybridoma was extracted, reverse transcribedand amplified by PCR using the degenerate signal sequence primer MHV4with heavy chain constant region primer MHCG1, or using the degeneratesignal sequence primer MKV4 with a kappa light chain constant regionprimer MKC. The resulting VH and VK sequences are shown.

FIG. 21: RNA from the JE12 hybridoma was extracted, reverse transcribedand amplified by PCR using a 5′ RACE PCR with heavy chain constantregion primer MHCG1, or using the signal sequence primer MKV1 with akappa light chain constant region primer MKC. The resulting VH and VKsequences are shown.

FIG. 22: RNA from the EH6 hybridoma was extracted, reverse transcribedand amplified by PCR using a 5′ RACE PCR with heavy chain constantregion primer MHCG2B, or using the signal sequence primer MKV with akappa light chain constant region primer MKC. The resulting VH and VKsequences are shown.

FIG. 23: RNA from the AG9 hybridoma was extracted, reverse transcribedand amplified by PCR using the degenerate signal sequence primer MHV7with heavy chain constant region primer MHCG1, or using a mix ofdegenerate signal sequence primers MKV1-11 with a kappa light chainconstant region primer MKC. The resulting VH and VK sequences are shown.

FIG. 24: RNA from the AH3 hybridoma was extracted, reverse transcribedand amplified by PCR using the degenerate signal sequence primer MHV7with heavy chain constant region primer MHCG2B, or using the signalsequence primer MKV1 with a kappa light chain constant region primerMKC. The resulting VH and VK sequences are shown.

FIG. 25: RNA from the DD9 hybridoma was extracted, reverse transcribedand amplified by PCR using a 5′ RACE PCR with heavy chain constantregion primer MHCG2A, or using the degenerate signal sequence primerMKV5 with a kappa light chain constant region primer MKC. The resultingVH and VK sequences are shown.

FIG. 26: RNA from the DH2 hybridoma was extracted, reverse transcribedand amplified by PCR using a 5′ RACE PCR with heavy chain constantregion primer MHCG2B, or using the degenerate signal sequence primerMKV45 with a kappa light chain constant region primer MKC. The resultingVH and VK sequences are shown.

FIG. 27: RNA from the DD6 hybridoma was extracted, reverse transcribedand amplified by PCR using a 5′ RACE PCR with heavy chain constantregion primer MHCG2B, or using a 5′ RACE PCR with a lambda light chainconstant region primer MLC. The resulting VH and VL sequences are shown.

FIG. 28: RNA from the IA12 hybridoma was extracted, reverse transcribedand amplified by PCR using the degenerate signal sequence primer MHV9with heavy chain constant region primer MHCG1, or using the degeneratesignal sequence primer CL14 with a kappa light chain constant regionprimer MKC. The resulting VH and VK sequences are shown.

FIG. 29. Dose response curves and IC50 values for enzymatic inhibitionof recombinant human TG2 by chimeric anti-TG2 antibodies, (a) cAB003,(b) cBB001, (c) cDC001, (d) cDD9001, (e) cDH001 and (f) the commercialTG2 antibody CUB7402. IC50 values are mean of 3 independent experiments.

FIG. 30. Dose response curves and IC50 values for enzymatic inhibitionof recombinant cynomogulus monkey TG2 by chimeric anti-TG2 antibodies(a) cDC001 and (b) the commercial TG2 antibody CUB7402.

FIG. 31. Dose response curves and IC50 values for enzymatic inhibitionof recombinant human TG2 by humanized anti-TG2 antibodies, (a) hBB001AA,(b) hBB001BB, (c) hAB005 and (d) hAB004.

FIG. 32. Dose response curves and IC50 values for enzymatic inhibitionof recombinant cynomogulus monkey TG2 by humanized anti-TG2 antibodies(a) hBB01AA and (b) hAB004.

FIG. 33. Dose response curves and IC50 values for enzymatic inhibitionof recombinant human TG2 by murine monoclonal anti-TG2 antibodies, (a)mAB003, (b) mBB001, (c) mDC001, (d) mDD9001, (e) mDH001 and (f) mDD6001.

FIG. 34—Binding ELISA of humanised versions of AB1 antibodies.

Supernatants from HEK293F cells co-transfected with differentcombinations of humanised AB1 light chains and AB1 heavy chain vectorswere assayed in an anti-human IgG ELISA to determine concentration andin an anti huTG2 ELISA. Each supernatant was assayed in triplicate andIC₅₀'s determined. The most potent combination was selected for furtherstudies and as the candidate humanised antibody.

FIG. 35. Dose response ELISA binding curves and EC50 data for antibodiesbinding to human TG2 (a) chimeric antibodies cDD9001, cDH001, cDC001,commercial TG2 antibody CUB7402 and isotype-matched control, (b)chimeric antibody cBB001 and isotype-matched control and (c) chimericantibody cAB003 and isotope-matched control.

FIG. 36. Dose response ELISA binding curves and EC50 data for antibodiesbinding to cynomogulus monkey TG2 (a) chimeric antibodies cDD9001,cDH001, cDC001, commercial TG2 antibody CUB7402 and isotype-matchedcontrol, (b) chimeric antibody cBB001 and isotype-matched control and(c) chimeric antibody cAB003 and isotope-matched control.

FIG. 37. Dose response ELISA binding curves and EC50 data for antibodiesbinding to human TG2 (a) humanized antibodies hBB001AA, HBB001BB,commercial TG2 antibody CUB7402 and isotype-matched control and (b)humanized antibody hAB004.

FIG. 38. Dose response ELISA binding curves and EC50 data for antibodiesbinding to cynomogulus monkey TG2 (a) humanized antibodies hBB001AA,HBB001BB, commercial TG2 antibody CUB7402 and isotype-matched controland (b) humanized antibody hAB004 and isotope-matched control.

FIG. 39: Humanised AB1 binding activity with extracellular TG2Inhibition of Extracellular TG2 activity produced by HK2 cells wasassayed using an ELISA measuring the incorporation of biotin cadaverineinto fibronectin. An exemplar curve showing the inhibition of TG2activity by humanised AB1 (hAB005) and the IC obtained is shown.

FIG. 40: Humanised BB7 binding activity with extracellular TG2.

Inhibition of Extracellular TG2 activity produced by HK2 cells wasassayed using an ELISA measuring the incorporation of biotin cadaverineinto fibronectin. An exemplar curve showing the inhibition of TG2activity by versions of humanised BB7 (hBB001AA and hBB001 BB) and theICs obtained is shown.

FIG. 41: Cytochalasin D, R281 and ZDON control scratch assay results andcommercial antibody CUB7402 scratch assay results.

Scratch wound assays were performed using WI-38 cell, after plating andovernight growth, cells were washed in media without serum and a scratchwound generated using an Essen Wound Maker. Media was removed andreplaces with 95 ul/well serum free media containing controls and testantibodies. The plate was placed in an Essen Incycte and the closure ofthe wound analysed using Incucyte software. Relative wound density wasplotted against time for the controls cytochalasin D, R281 and Z-Don(panel A) and the commercial antibody CUB7402 and cytochalasin (panelB).

FIG. 42: Humanised BB7 scratch assay results.

Scratch wound assays were performed using WI-38 cell, after plating andovernight growth, cells were washed in media without serum and a scratchwound generated using an Essen Wound Maker. Media was removed andreplaces with 95 ul/well serum free media containing controls and testantibodies. The plate was placed in an Essen Incycte and the closure ofthe wound analysed using Incucyte software. Relative wound density wasplotted against time for the humanised hBB001 AA and the controlcytochalasin D (panel A) and hBB001BB and the control cytochalasin D(panel B).

FIG. 43: Humanised AB1 scratch assay results.

Scratch wound assays were performed using WI-38 cell, after plating andovernight growth, cells were washed in media without serum and a scratchwound generated using an Essen Wound Maker. Media was removed andreplaces with 95 ul/well serum free media containing controls and testantibodies. The plate was placed in an Essen Incycte and the closure ofthe wound analysed using Incucyte software. Relative wound density wasplotted against time for the humanised hAB005 and the controlcytochalasin D

FIG. 44: Chimeric DC1 scratch assay results.

Scratch wound assays were performed using WI-38 cell, after plating andovernight growth, cells were washed in media without serum and a scratchwound generated using an Essen Wound Maker. Media was removed andreplaces with 95 ul/well serum free media containing controls and testantibodies. The plate was placed in an Essen Incycte and the closure ofthe wound analysed using Incucyte software. Relative wound density wasplotted against time for the chimeric antibody cDC001 and the controlcytochalasin D

FIG. 45: Human TG2 binding to cAB003 immobilised antibody by Biacore.The association phases of human TG2 injections over the cAB003-coatedbiosensor at 25, 50, 100 and 200 nM, including in duplicate at 50 nM,are shown on the left. From the same experiment, two long dissociationphases were collected, as shown on the right. Fits are shown as solidblack lines and the results are shown in Table 25.

FIG. 46: Cynomolgus monkey TG2 binding to hAB004 immobilised antibody byBiacore. The association phases of cynomolgus monkey TG2 injections overthe hAB004-coated biosensor at 25, 50, 100, 200 and 400 nM, including induplicate at 50 nM, are shown on the left. From the same experiment, twolong dissociation phases were collected, as shown on the right. Fits areshown as solid black lines and the results are shown in Table 26.

FIG. 47: Human TG2 binding to cDH001 immobilised antibody by Biacore inthe absence of calcium. The association phases of human TG2 injectionsover the cDH001-coated biosensor at 25, 50, 100 and 200 nM, including induplicate at 50 nM, are shown on the left. From the same experiment, twolong dissociation phases were collected, as shown on the right. Fits areshown as solid black lines and the results are shown in Table 25.

EXAMPLE 1: DEVELOPING A TG2 INHIBITORY ANTIBODY SUITABLE FOR THERAPEUTICUSE IN MAN WITH THE IDENTIFICATION OF 3 SPECIFIC INHIBITORY EPITOPES

Transglutaminase type 2 (TG2) catalyses the formation of anε-(γ-glutamyl)-lysine isopeptide bond between adjacent peptides orproteins including those of the extracellular matrix (ECM). Elevatedextracellular TG2 leads to accelerated ECM deposition and reducedclearance that underlies tissue scarring and fibrosis. It also is linkedto celiac disease, neurodegenerative disorders and some cancers. Whilenumerous compounds have been developed that inhibit transglutaminases,none of these are specific to TG2, inhibiting all transglutaminases tosome extent. While these have allowed proof of concept studies for TG2'srole in these pathologies, the lack of isoform specificity has preventedtheir application in man. To address this, we set out to develop a highaffinity TG2 specific antibody that would inhibit only TG2 activity.

A recombinant protein encompassing amino acids 143 to 473 of the humanTG2 core was produced in Escherichia coli, re-folded and 100 μg injectedinto 4 mice with boosts at 2, 5, 7, and 10 weeks. Spleens were recovered4 days after the final boost and splenocytes fused to Sp2/0-Ag-14myeloma cells. Seventy-five hybridoma supernatants showed specificity toTG2. These hybridoma supernatants were screened for their ability toinhibit TG2 activity in a putrescine incorporation assay containing 100μg of TG2. Ten TG2 specific supernatants were inhibitory. These weresubsequently double cloned. Using phage display to screen a TG2 fragmentlibrary, each antibody was mapped to a precise epitope in the TG2 coredomain and 3 distinct inhibitory epitopes determined. The amount ofantibody to reduce the activity from 100 ng of TG2 by 50% wasdetermined.

The 2 most effective antibodies, AB1 and DC1 bound to amino acids 304 to327 and had an IC₅₀ of 1.1×10⁻⁵ mg/ml IgG per ng of recombinant TG2.Application of AB1 & DC1 was able to inhibit TG2 successfully in humanHep2G cells and extracellular TG2 in human HK-2 cells when applied tothe culture media.

Thus, immunisation of mice with the TG2 core domain surprisingly enabledthe generation of monoclonal antibodies that target previouslyunreported epitopes within the catalytic core. These antibodies arespecific, inhibit TG2 activity effectively and are suitable for in vivoapplication.

Materials and Methods Transglutaminase 2 Catalytic Core DomainProduction

The catalytic core domain of human TG2 (residues Cys143-Met 473 of TG2)was expressed, refolded and purified to permit immunisation in mice. Thecatalytic core domain (PCR sense primer GCG CGC GCT AGC TGC CCA GCG GATGCT GTG TAC CTG GAC, anti-sense GCG CGC AAG CTT CAT CCC TGT CTC CTC CTTCTC GGC CAG) was cloned into the expression vector pET21a(+) andexpressed as insoluble inclusion bodies in E. coli strainBL21-CodonPlus(DE3)-RIPL (Agilent Technologies). In brief, 50 μl ofcompetent BL21 (DE3) pLysS cells were transformed with 1 μl of theexpression plasmid (30 ng/μl) and plated onto LB agar plates containingthe selective antibiotics (100 μg/ml ampicillin, 34 μg/mlchloramphenicol) and 1% glucose and incubated overnight at 37° C. Asingle colony was picked to seed 10 ml of fresh LB medium containing 100μg/ml ampicillin, 34 μg/ml chloramphenicol and 1% glucose in shakingincubator at 37° C. and at 200 rpm. After overnight growth, cultureswere transferred in 100 ml 2×YT media with 1% glucose and grown to anOD_(600nm) of 0.8 and then transferred to 1 L 2×YT medium until theOD_(600nm) reached 0.8 again. After 4 hours induction under 1 mM IPTG tostimulate expression, pelleted and bacteria were lysed by sonication inbuffer A (10 mM Tris; 1 mM EDTA; 10 mM DTT; 1 mM PMSF; 0.5 mg/mllysozyme protease inhibitor tablets (Roche), pH 8.0). Inclusion bodieswere harvested by centrifugation at 40,000×g and washed three times inwash buffer B (50 mM Tris; 1 mM EDTA; 10 mM DTT; 2% sodium deoxycholate,pH 8.0) before a final wash in deionised water.

Inclusion bodies were solubilised in 3.5 mls of resolubilisation buffer(40 mM Tris-HCl, 8 M urea, and 10 mM DTT pH12) and refolded over aperiod of 16 hours in refolding buffer (40 mM Tris HCl; 150 mM NaCl; 20%glycerol; 5 mM cysteine; 0.5 mM cystine pH 8) at 4° C. in the dark.

The resolubilised inclusion bodies were loaded onto a 1 ml Nickelcolumn. Briefly, the column was pre-equilibrated with binding buffer (40mM Tris; 300 mM NaCl; 10 mM Imidazole) and the inclusion bodies applied.The column was extensively washed (40 mM Tris; 300 mM NaCl; 30 mMimidazole). The recombinant protein was eluted by high concentrationimidazole buffer (40 mM Tris; 300 mM NaCl; 300 mM imidazole). Elutedprotein containing fractions were pooled and dialysed overnight againstan appropriate buffer (40 mM Tris; 300 mM NaCl pH 8). Protein wasassessed using the Bradford protein assay

HepG2 Cell Culture & Lysates

HepG2 cells were kindly supplied by Richard Ross (University ofSheffield). Cells were routinely grown at 37° C. in a 95% humidifiedatmosphere of 5% CO₂ in DMEM/4.5 g per litre glucose supplemented with10% foetal calf serum (FCS), 100 IU penicillin and 100 μg/mlstreptomycin, 2 mM I-glutamine (all GIBCO). Two million cells wereseeded on a 10 cm dishes and grown for 48 hours. Cells were lysed in 250μl of STE buffer (0.32M sucrose, 5 mM Tris, 1 mM EDTA containingprotease inhibitors Phenylmethylsulphonyl fluoride (1 mM), benzamidine(5 mM), and leupeptin (10 μg/ml) and sonicated on ice to produce a celllysate usable in TG2 activity assay.

Human Kidney 2 (HK2) Cells:

HK-2 cells (kidney proximal tubular epithelium) were purchased from theEuropean cell culture collection at passage 3. Cells were routinelygrown at 37° C. in a 95% humidified atmosphere of 5% CO₂ in keratinocyteserum free medium (KSFM, Gibco 17005-042) with L-glutamine supplementedwith recombinant EGF (0.1-0.2 ng/ml) and bovine pituitary extract (20-30ug/ml). For passage, media was removed and washed once with 1×PBS beforetrypsinising with 1 ml of 0.25% trypsin/EDTA (T75 flask) for 1 minute at37° C. Cells were resuspended in 10 mls of KSFM and centrifuged at 400 gfor 1 minute. Media was removed and cells plated in KSFM (1:3 to 1:5split is normal). Cells were used experimentally at passages 5-14. Cellstypically grew well to 95% confluence.

Coomassie Staining and Western Blotting

The purity of recombinant proteins was checked by running 5 μg of therecovered protein on a 10% (w/v) polyacrylamide denaturing gel andstaining with Coomassie Brilliant Blue R staining solution (Sigma).

Confirmation of TG2 core protein synthesis as well as TG2 and TG2 corereactivity levels following immunisation were all measured by westernblotting. Recombinant proteins (10 to 80 ng) were loaded on a 10% (w/v)polyacrylamide denaturing or non-denaturing gel as required andtransferred onto PVDF membranes (Transblot SD, Biorad, UK) for one hourat 100 V. Membranes were blocked overnight at 4° C. with 3% (w/v) BSA inTBS/0.1% (v/v) Tween 20. The membranes were then washed and probed withmonoclonal mouse anti-transglutaminase antibodies in TBS/Tweencontaining 1% BSA. For proof of recombinant TG2 core protein and as apositive control for antibody screening the commercial antibody Cub7402(neomarkers) was used at a 1:1000 dilution. Binding of primary antibodywas detected with the anti-mouse gamma-chain-HRP linked secondaryantibody (Sigma, Poole, UK). Bands were visualised using ECLchemiluminescent detection system (Amersham, UK).

Mouse Immunisation and Fusion

Each mouse was immunised with a mixture of 50 μg of antigen (made up toa volume of 50 μl with sterile PBS) and 50 μl of complete Freund'sadjuvant. Four (8-12 week old) BALB/C mice were injected. Two boostimmunisations were carried out (day 14 and day 35) using the sameprocedure with the exception that incomplete Freund's adjuvant was usedfor these injections. At day 45, test bleeds were taken from all animalsand assessed for reactivity to TG2 by ELISA.

The two best responders were further boosted by injection of 100 μg ofcore protein (in PBS) again mixed with incomplete Freuds Adjuvant at 10weeks, and 4 days later the animals were sacrificed for splenocyterecovery and fusion with Sp2/0-Ag-14 myeloma cells. From this fusion,approximately 1000 wells were screened for reactivity to TG2 protein byELISA.

Screening for TG2 Specificity

Conditioned medium or purified IgG were tested for reactivity totransglutaminase family members. The ability of each to bind to eachtransglutaminase (TG1, TG2, TG3, TG5, TG7 and Factor XIIIa; all Zedira)was determined using a plate binding assay. Microtiter plates (Costar,Cambridge, UK) were coated with recombinant TG (Zedira, Darmstadt,Germany) in 50 μl of 0.1 M bicarbonate/carbonate buffer (pH 9.6)overnight at 4° C. Plates were blocked for 2 h at 37° C. with 200 μl PBScontaining 3% w/v BSA. Plates were washed three times with PBScontaining 0.05% Tween 20 (washing buffer) and 100 μl of dilutedconditioned medium (dilution 1:5 to 1:20) or purified anti-TG2 catalyticcore mAbs was added. Plates were incubated for a further 1 h at roomtemperature. The washing step was repeated and anti-mouse gammachain-horseradish peroxidase (1:5000) in PBS containing 0.05% Tween 20(v:v) and 1% BSA (w:v) (Sigma, Poole UK) was added for 1 h. After eightwashes, binding was revealed with 50 μl of3,3′,5,5′-tetramethylbenzidine substrate. The reaction was stopped byadding 25 μl of 0.1 M H₂SO₄ and the absorbance at 450 nm was determined.

Screening for TG2 Inhibition

TG activity is measured by the Ca²⁺ dependent incorporation of³H-putrescine into N′,N′-dimethylcasein. Recombinant human TG2 (100 ng)was pre-incubated for twenty minutes at room temperature with the testsample (conditioned medium or purified IgG) before starting thereaction. Twenty-five μl of reaction mix (5 μl of 25 mM CaCl₂, 5 μl of40 mM dithiothreitol, 5 μl ³H-putrescine mix, and 10 μl 25 mg/ml of N,N′dimethylcasein (replace 25 mM CaCl₂ with 100 mM EDTA for a non-enzymaticcontrol) was added to start the reaction and the samples incubated at37° C. for up to 1 hour. Aliquots of 10 μl were spotted onto a strip of3 MM Whatman filter paper and plunged immediately into ice-cold 10%trichloroacetic acid (TCA) in order to precipitate the cross-linkedproteins typically at time 0, 10, 30 and 60 minutes into the reaction.After three extensive washes in ice-cold 5% TCA followed by 3 rinseswith ice-cold 95% ethanol, the air dried filter was counted in 2 ml ofscintillation fluid (Ultima Gold Packard, Perkin Elmer). The rate ofreaction was calculated. 1 TG unit is equivalent to the incorporation of1 nmol of putrescine per hour at 37° C.

The same protocol was used to assess TG inhibition in cell lysates byreplacing the 25 μl of recombinant protein with 25 μl of cell lysate.

Hybridoma Cloning & Purification of Antibodies from Conditioned Medium

Monoclonal antibody isolation was undertaken from the cloned inhibitoryhybridomas. Initially identified hybridoma wells were doubly cloned by alimiting dilution process (to ensure stability and clonality) accordingto conventional methods (Loirat M J et al, 1992) with sub-clones testedas described by ELISA and activity screens. Selected antibody producingclones were expanded in 25 and 75 cm² flasks and fed with serum freemedium (Hyclone, Fisher Scientific, Loughborough, UK). As cells wereexpanded, conditioned medium was collected for IgG purification usingaffinity chromatography on protein G column (Amersham Life Sciences).The conditioned medium was diluted in an equal volume of 10 mM sodiumphosphate, pH 7.25, and applied to the protein G column at a flow rateof 1.0 to 2.0 ml/min. The column was extensively washed with 10 columnvolumes of the same buffer. Bound antibody was eluted in glycinesolution (0.1M; pH 2.7) and neutralised by 0.15% volumes of 1M Tris/HClpH 9. Samples were dialysed against 1000 volumes of phosphate buffersaline solution for 24 hours with 2 buffer changes.

Phage Display Mapping of Antibody Epitopes

The full length coding sequence of human TG2 was amplified by polymerasechain reaction using the following primers; TG2-FL-1 5′ATGGCCGAGGAGCTGGTCTTAGAGA 3′ and TG2-FL-2 5′ GGCGGGGCCAATGATGACATTCCGGA3′. The approximately 2 kb amplification product was purified usingQuiagen PCR cleanup kit (Qiagen) and digested into random fragmentsusing RQ DNAse I (Promega). The RQ DNAse reaction was treated withKlenow fragment of DNA polymerase I and T4 DNA polymerase to generateblunt-ended fragments. These were purified by gel electrophoresis, andfragments in the range of 50-150 bp extracted using Qiagen gel recoverykit (Qiagen, Crawley UK).

A phage display vector was digested with EcoRV, treated with alkalinephosphatase and purified by gel electrophoresis and the Qiagen gelrecovery kit. 100 ng of purified vector was ligated to 15 ng of preparedblunt fragments of human TG2 cDNA. The resultant ligation waselectroporated into XL1-Blue electrocompetent cells (AgilentTechnologies) and the fragment library rescued with VCSM13 helper phage(Agilent). Phage particles were precipitated with 2% glucose and 4% PEG6000 and resuspended in PBS 0.1 Tween 20 (v:v) 1% BSA (w:v).

Epitope mapping was carried out using the following procedure. ELISAwells were coated overnight at 4° C. with 30 μg of monoclonal antibodyin 100 μl of coating buffer. The coated well was washed with PBS/Tweenand blocked with 400 μl of 3% BSA in PBS (w:v) for 1 h at roomtemperature. Approximately 10¹⁰ phage particles (100 μl) were added tothe blocked well and incubated at room temperature for 1 h. The well waswashed 8 times with 400 μl PBS/0.5% Tween (v:v) and adherent phageeluted with 0.2 M glycine pH 2.2. Eluted phage were used to infect 1 mlof XL1-Blue host and samples plated onto LB agar (60 μg/ml ampicillin,15 μg/ml tetracycline), the remaining host was added to 100 ml LB media(60 μg/ml ampicillin, 15 μg/ml tetracycline) and grown overnight at 37°C. in a shaking incubator at 200 rpm to generate the enriched library ofselected fragments. This enrichment process was repeated 5 times andrandom colonies from the final round were selected for sequencing.

Determining the Sequence of the Antibody VL Region Primers

Heavy chain sense primers—A pair of highly degenerate FR1 primers, MH1and MH2 (Wang et al 2000), were combined with 3 constant region primersto amplify VH genes.

MH1 5′ CGCGCGCTCGAGSARGTNMAGCTGSAGTC 3′ MH25′CGCGCGCTCGAGSARGTNMAGCTGSAGSAGTC 3′ Mouse-G15′ AGGCGCAGTACTACAATCCCTGGGCACAATTTTCTTGTCCACC 3′ Mouse-G2a5′ AGGCGCAGTACTACAGGGCTTGATTGTGGGCCCTCTGGG 3′ Mouse-G2b5′ AGGCGCAGTACTACAGGGGTTGATTGTTGAAATGGGCCCG 3′ Kappa primers VK15′ CGCTGCGAGCTCGATATTGTGATGACBCAGDC 3′ VK25′ CGCTGCGAGCTCGAGRTTKTGATGACCCARAC 3′ VK35′ CGCTGCGAGCTCGAAAATGTGCTCACCCAGTC 3′ VK45′ CGCTGCGAGCTCGAYATTGTGATGACACAGTC 3′ VK55′ CGCTGCGAGCTCGACATCCAGATGACACAGAC 3′ VK65′ CGCTGCGAGCTCGAYATTGTGCTSACYCARTC 3′ VK75′ CGCTGCGAGCTCGACATCCAGATGACYCARTC 3′ VK85′ CGCTGCGAGCTCCAAATTGTTCTCACCCAGTC 3′ K-CONST5′ GCGCCGTCTAGAATTAACACTCATTCCTGTTGAA 3′

Total RNA was extracted from monoclonal hybridoma cells (˜10⁵ cells)using Trizol (GIBCO) according to the manufacturer's protocol andquantified by A_(260nm). cDNA was synthesised using ImProm II reversetranscriptase (Promega) and random hexamer primers. The reaction mix wasas follows; 1 μg total RNA, 0.1 μg oligo (dN)₆, 12 μl ImProm II buffer,1 μl 10 mM dNTPs (Promega), 8 μl 25 mM MgCl₂, 4 μl ImProm II reversetranscriptase (Promega), DEPC-treated H₂O up to total reaction volume of60 μl. The RNA and random primer mix was heated to 70° C. for 10 min andthen placed on ice. The remaining reaction components were added andthen incubated at 20° C. for 10 min, then at 40° C. for a further 40min.

Amplification of VH and VK genes was carried out with GoTaq polymerase(Promega). Each 50 μl reaction contained the following; cDNA 2 μl, 20pmol sense and antisense primers, 10 μl GoTaq reaction buffer, 1 μl 10mM dNTPs, 5 μl 25 mM MgCl₂, 2.5 u GoTaq polymerase, H₂O to a finalvolume of 50 μl. Reactions were cycled 35 times using the followingconditions: initial denature 95° C. 2 min; denature 94° C. 1 min, anneal56° C. 1 min, extension 72° C. 1 min. PCR products were analysed by gelelectrophoresis and cloned using the TOPO TA cloning kit (Invitrogen).Random minipreps of heavy and light chain PCR products were selected forsequencing.

Measurement of Extracellular TG Activity

Extracellular TG activity was measured by modified cell ELISA. HK-2epithelial cells were harvested using 0.1M EDTA or 0.25% trypsin/EDTAand plated at a density of 8×10⁴ cells/well in serum free medium onto a96 well plate that had been coated overnight with 100 μl/well offibronectin (5 μg/ml in 50 mM Tris-HCl pH 7.4) (Sigma, Poole UK). Cellswere allowed to attach for 2.5 h at 37° C. in the presence of the 0.1 mMbiotin cadaverine [N-(5 amino pentyl biotinamide) trifluoroacetic acid](Molecular Probes, Eugene Oreg., USA). Plates were washed twice with 3mM EDTA/PBS and cells removed with 0.1% (w/v) deoxycholate in 5 mMEDTA/PBS. The supernatant was collected and used for proteindetermination. Plates were washed with 50 mMTris-HCl and incorporatedbiotin cadaverine revealed using 1:5000 extravidin HRP (Sigma, Poole,UK) for 1 h at room temperature followed by a TMB(3,3′,5,5′-tetramethylbenzidine) substrate. The reaction was stoppedwith 50 μl 2.5 M H2SO4 and the absorbance read at 450 nm.

Measurement of Collagen Levels by Radiolabelling

Cells were seeded at a density of 3.75×10⁶/10 cm² Petri dish or1×10⁶/well of a 6 well plate. ECM collagen was assessed by labellingwith 20 iCi of ^(3,4)H proline (1.0 mCi/ml, ICN). Labelling wasperformed for 72 h under standard cell culture conditions. Followinglabelling, the media was removed, cells washed with PBS and removed with2 ml of 0.25 M ammonium hydroxide in 50 mM Tris pH 7.4 at 37° C. for 10min. The soluble fraction was collected and protein concentrationdetermined using the bicinchoninic acid (BCA) assay. The dishes werewashed extensively with increasing volumes of PBS before the ECM wassolubilised with 2 ml of 2.5% (w/v) SDS in 50 mM Tris pH 6.8. The dishwas scraped to ensure complete removal of the ECM and 200 il wasmeasured for radioactivity in a beta scintillation counter. Counts werecorrected per mg of solubilised cell protein and expressed as apercentage of the mean control value.

Generation of Recombinant Ratified Quark IgG

For experimental purposes a human-rat chimeric antibody from thesequence of a ‘human’ single-chain Fv of an antibody against humantype-II transglutaminase was generated. The antibody is called QPCDTGII(shortened to QCT), and the sequences of the variable regions areavailable in WO 2006/100679A2.

A rat γ2a subclass was selected for the heavy chain constant regions,removing the glycosylation site to reduce the chance of an ADCC reactionin the rat test animals. The selected rat constant region for the heavychain was 013593 (Bruggemann, M. Gene 74: 473-482 (1988); Bruggemann,M., Free, J., Diamond, A., Howard, J., Cobbold, S. and Waldmann, H.Proc. Natl. Acad. Sci USA 83: 6075-6079 (1986)) from the Kabat database(Kabat, E. A., Wu, T. T., Perry, H. M., Gottesman, K. S., and Foeller,C. Sequences of Proteins of Immunological Interest. (NIH NationalTechnical Information Service, 1991)). That for the kappa light chainwas 013718 (Sheppard, H. W. and Gutman, G. A. Proc. Natl. Acad. Sci. USA78: 7064-7068 (1981)) from Kabat.

In brief, heavy chain and kappa chain coding sequences were generated byDNA synthesis (codon usage was adapted to a mammalian codon bias).

The heavy chain gene synthesis product was amplified by PCR using theprimers QCT_HindIII and QCT_H_rev. The PCR-product was cut with HindIIIand NgoMIV and ligated into MRCT expression vector. Clones of competentDH5α bacteria chemically transformed by the ligation product werePCR-screened using the primers HCMVi and rat_gamma1. Three clonesgenerating a PCR product of the predicted size were sequenced.

The kappa chain gene synthesis product was amplified by PCR using theprimers QCT_HindIII and QCT_L_rev. The PCR-product was cut with HindIIIand PpuMI and ligated into the expression vector pKN100. Clones ofcompetent DH5α bacteria chemically transformed by the ligation productwere PCR-screened using the primers HCMVi and rat_kappa. Three clonesgenerating a PCR product of the predicted size were sequenced.

A double insert expression vector coding for both Heavy and kappa chainswas generated and transfected into HEK293T cells. Cell culturesupernatant from two large scale HEK293T transfections was pooled andaffinity purified on a 1 ml Protein L-agarose column using an ÄKTAExplorer chromatography system, in accordance with the manufacturer'sprotocols. A single OD 280 nm peak eluted with IgG Elution Buffer, andwas dialysed against two changes of PBS. This was assayed both by UVabsorption at 280 nm, and by rat IgG_(2a) ELISA. The total yield wasapproximately 700 μg (by OD_(260nm)); 303.5 μg (by ELISA).

Humanisation of AB1 Antibody

Human VH and VK cDNA Databases

The protein sequences of human and mouse immunoglobulins from theInternational Immunogenetics Database 2009¹⁰¹ and the Kabat DatabaseRelease 5 of Sequences of Proteins of Immunological Interest (lastupdate 17 Nov. 1999)¹⁰² were used to compile a database of humanimmunoglobulin sequences in a Kabat alignment. Our database contains10,606 VH and 2,910 VK sequences.

Molecular Model of AB1

A homology model of the mouse antibody AB1 variable regions has beencalculated using the Modeller program¹⁰³ run in automatic mode. Theatomic coordinates of 1 MQK.pdb, 3LIZ.pdb and 1MQK.pdb were the highestidentity sequence templates for the Interface, VL and VH respectively asdetermined by Blast analysis of the Accelrys antibody pdb structuresdatabase. These templates were used to generate 20 initial models, thebest of which was refined by modeling each CDR loop with its 3 best looptemplates.

hAB1 Framework Selection

The sequence analysis program, gibsSR, was used to interrogate the humanVH and VK databases with the AB1 VHc, VKc and VKc₁ protein sequencesusing various selection criteria. FW residues within 5 Å of a CDRresidue (Kabat definition) in the homology model of mouse antibody AB1,were identified, and designated as the “5 Å Proximity” residues.

AF06220 was chosen as the FW on which to base the initial humanised AB1VHc construct. Table 1 shows the alignment and residue identity ofAF06220 to murine Ab1. Table 2 shows the 5 Å proximity envelope of thesequences. AF062260 has only 1 somatic mutation away from its germlineVH gene Z12347 (Table 3).

AY247656 was chosen as the FW on which to base the initial humanised AB1VKc construct. The alignment and residue identity to murine AB1 areshown in Table 4; Table 5 shows the 5 Å proximity envelope of thesequences. The sequence shows 5 somatic mutations from its germline VKgene X93620 (Table 6).

AF193851 was chosen as the FW on which to base the initial AB1 VKc₁construct. The alignment and residue identity to murine AB1 are shown inTable 7. Table 8 shows the 5 Å proximity envelope of the sequences. Thesequence shows no somatic mutations from its germline VK gene J00248(Table 9).

Binding ELISA

HEK 293F cells were co-transfected with combinations of differenthumanised light chain vectors in association with different humanisedheavy chain vectors. Recombinant human TG2 was used to measure antibodybinding by ELISA. The results indicated that the Heavy Chain version RHA(Table 10), in combination with either Light Chain versions RKE and RKJ(Table 11) (representing the different Light Chain versions humanised)showed optimal binding (FIG. 16).

Heavy Chain version RHA is an un-modified graft of the mouse CDR regionsof the AB1 antibody onto the Human donor sequence. However, both LightChain versions RKE and RKJ, have the same single 5 Å proximity residebackmutation, F72 (Kabat numbering—shown in green). This backmutationlies outside the Vernier¹⁰⁴, Canonical¹⁰⁵ or Interface¹⁰⁶ residues (seeTable 11).

-   101. Lefranc, M. P. IMGT, the international ImMunoGeneTics    Database®. Nucleic Acids Res. 31, 307-310 (2003).-   102. Kabat, E. A., Wu, T. T., Perry, H. M., Gottesman, K. S. &    Foeller, C. Sequences of Proteins of Immunological Interest. NIH    National Technical Information Service, (1991).-   103. Eswar, N. et al. Comparative protein structure modeling using    Modeller. Curr. Protoc. Bioinformatics. Chapter 5: Unit 5.6., Unit    (2006).-   104. Foote, J. & Winter, G. (1992). Antibody framework residues    affecting the conformation of the hypervariable loops. J Mol. Biol.    224, 487-499.-   105. Morea, V., Lesk, A. M. & Tramontano, A. (2000). Antibody    modeling: implications for engineering and design. Methods 20,    267-279.-   106. Chothia, C., Novotny, J., Bruccoleri, R. & Karplus, M. (1985).    Domain association in immunoglobulin molecules. The packing of    variable domains. J Mol. Biol. 186, 651-663.

TABLE 2 5Å Proximity Residues AB_VHc EVQLCAFTLSWVRWVARFTISRNLYCAKWGAF062260 ........F......S..............

TABLE 5 5Å Proximity Residues AB_VKc EIVLTQTCWFTLIYGVPFSGSGSGQDFFYCFGAY247656 .........YL.............T..T....

TABLE 8 5Å Proximity Residues AB_VKc DIQMTQTCWFTLIYGVPFSGSGSGQDFFYCFG

..........S.............T..T....

Results Generation of Rh TG2 Core Protein.

To force the generation of antibodies that would be more likely totarget epitopes critical for TG2 activity, rather than favoured sites onthe TG2 molecule, we immunised mice with the TG2 catalytic core ratherthan the full-length TG2 molecule. To generate the recombinant TG2domain, a PCR construct was generated running from bases 329 to 1419 andinserted into the Pet21+(a) vector (FIG. 1A). Insertion and expressionof this vector in BL21-CodonPlus (DE3)-RIPL bacteria resulted in thegeneration of an insoluble protein spanning amino acids 143 to 473encompassing the entire catalytic core. This protein was solubilised andrefolded in 40 mM Tris HCl; 150 mM NaCl; 20% glycerol; 5 mM cysteine;0.5 mM cystine pH 8. 10 ng of this was run on a non-reducingpolyacrylamide gel, western blotted and immunoprobed with CUB7402. Aclear band was visible at 37 kDa which is consistent with the predictedsize of the TG2 core (FIG. 1B). Larger bands were also immunoreactive toCUB7402 which most likely represent aggregates of the core protein asthese were not present when a reducing gel was run (data not shown).

Immunisation and Fusion.

Four mice were immunised with 50 μg of rhTG2 core. At approximately fiveand nine weeks post immunisation, a serum sample was taken from eachmouse and tested for reactivity against rh TG2 by ELISA using a serialdilution of the serum. All mice showed a strong immune reaction to rhTG2core, even at the highest dilution used (1:51 000) (FIG. 2A). To confirmthe antibodies would also recognise the full-length TG2, rhTG2 and rhTG2core protein were run on a non-denaturing gel, western blotted andimmunoprobed with a 1 in 1000 dilution of mouse serum (FIG. 2B). Themouse with the strongest reactivity (mouse C) against both proteins wasboosted and splenocytes recovered for fusion using the University ofSheffield's Hybridoma service, Bioserv.

Selection of Positive Hybridoma and Cloning.

Out of 400 hybridoma wells selected by Bioserv as highest positives,supernatants from 109 showed persistent reactivity to TG2, however only34 did not react with other key TG family members when tested in ELISA(representative examples shown in FIG. 3A). Those that were specific toTG2 had the supernatant screened by ³H-putrescine incorporation assayfor the ability to inhibit TG2 activity resulting from 100 ng of TG2(FIG. 4). This initial screen indicated that 10 hybridoma supernatantswere able to inhibit TG2 activity (AB1; DC1; BB7; EH6; DH2; DD9; JE12;AG9; AH3; DF4). Nine of the ten were successfully cloned by limiteddilution. For the clone DF4, although clones were isolated post-cloning,they did not appear to be inhibitory. Post cloning, IgG was purifiedfrom each cloned hybridoma and retested for selective reactivity to TG2(FIG. 3B).

TG2 Inhibitory Potential.

Each cloned hybridoma had its IgG tested for TG2 inhibitory activityagainst human, rat and mouse TG2 and the IC₅₀ calculated based on theamount of IgG required to inhibit 1 ng of TG2. There was approximately a12 fold range in IC₅₀ values against human TG2 ranging from the mosteffective AB-1 at 1.1×10⁻⁵ mg/ml of IgG to the least effective JE12 at12.3 1.1×10⁻³ mg/ml of IgG (FIG. 9; Table 1). Interestingly we were onlyable to determine an IC₅₀ for 4 antibodies (DH2, DD9, EH6 and BB7)against rat TG2 with the best, DH2 having a IC₅₀ of 2.23×10⁻⁴ mg/ml ofIgG being some 6 fold less active than against human TG2 andcomparatively 38 fold less active that the best AB-1 inhibitor againstTG2 (FIG. 9; Table 1). None of the inhibitory antibodies were able toinhibit mouse TG2, probably due to immune tolerance.

Mapping the Epitopes of Inhibitory Antibodies

To establish which epitopes in TG2 were immunologically unique to TG2while inhibitory, as well as establishing if these 10 antibodies weretargeting the same or different sites, each antibody was mapped usingphage display. A TG2 phage library was constructed and panned againsteach mAb. The epitope was then determined by consensus sequencing of thebinding phages.

AB1, AG1, AH1, BB7, DC1, EH6 and JE12 all appeared to bind in whole orpart to a single epitope (FIG. 5) which encompasses amino acids 304 to326 and appears to sit in front of active site within a substratebinding pocket (FIG. 6). This region we termed the AB-1 site and calledantibodies targeting this site Group 1 antibodies.

DF4 uniquely targeted a sequence running from amino acid 351 to 365(FIG. 5) which runs from front to rear of core encompassing Asp 358 inthe catalytic triad (FIG. 6). This we termed Group 2.

DH2 and DD9 bound to a sequence spanning amino acids 450 to 467 (FIG.5). These Group 3 antibodies bind to a region at the rear of core nearthe junction with β barrel-1, that we termed the DH2 site. The epitopeencompasses a putative calcium binding site (FIG. 6).

Antibody Sequencing

In order to establish the variable light chain sequence for eachantibody, RNA from each inhibitory hybridoma was extracted, reversetranscribed and amplified by PCR using a pair of highly degenerate FR1primers, MH1 and MH2 primers being combined with 3 constant regionprimers to amplify VH genes.

The resulting VH and VK sequences are shown in FIG. 7 for AB1.

The Ability of AB1 to Inhibit TG2 Activity in a Protein Mixture InVitro.

The most potent inhibitory antibody against recombinant TG2 is AB1. Tobe of value therapeutically, it must be able to not only inhibit TG2activity in a pure solution, but also in a complex protein solution andnot associate in anyway with other proteins. To test this, a homogenateof the human hepatocyte cell line HepG2 was prepared. Application of 0.5μg of AB1 was able to inhibit 70% of the TG2 activity (FIG. 8A). HoweverBB7 produced a significantly better inhibition knocking down 90% of theTG2 activity. Immunoprobing 25 μg of this lysate with AB1 showed no offtarget association with a single immunoreactive band at a sizecorresponding to TG2 (FIG. 8B).

The Ability of AB1 and DC1 to Inhibit Extracellular TG2 Activity.

To assess if these antibodies could inhibit TG2 activity in a cellsystem. AB1 (FIG. 10a ) and DC1 (FIG. 10b ) were applied to human kidney2 (HK-2) tubular epithelial cells in culture and extracellular TGactivity assayed using the biotin cadaverine assay. AB1 was able toachieve a 60% inhibition and DC1 a 55% inhibition of activity whenapplied at 4 ng/ul in the culture media which was comparable to thechemical pan TG inhibitor 1,3-Dimethyl-2-[(2-oxo-propyl)thio]imidazoliumchloride applied at 400 uM.

Comparison of Antibody AB1 with Other Known Inhibitory Antibodies

To test the effectiveness of AB1 in comparison to other known TG2inhibitory antibodies both fab fragments (FIGS. 11,12) and full IgG(FIGS. 13, 14) of an antibody as described by Quark biotechnology inpatent application number WO2006/100679 were tested to inhibit TG2activity in the ³H Putrescine incorporation assay. The activity from 100ng of human TG2 could be inhibited by 60 to 80% by 500 ng of AB1. Incomparison neither the fab fragment or the full IgG of the Quarkantibody could inhibit TG2 significantly in this assay.

DISCUSSION

There is a clear need to validate TG2 as a therapeutic target in manacross a range of diseases where experimental studies have suggested itsinvolvement. These include tissue scarring, celiac disease,neurodegenerative diseases and chemo-resistance in some cancers.Limiting this has been the lack of truly TG2 specific compounds that canselectively inhibit TG2 activity in man.

In this study we have for the first time immunised mice with a fragmentof TG2 with the aim of being able to isolate a wider range of anti TG2antibodies against the enzyme's catalytic core in the search of aninhibitory epitope. This elicited a good immune response with antibodiesrecognising both the rhTG2 core and native TG2 but no other TG.

10 of the antibodies isolated showed inhibitory activity. These weresubsequently mapped to 3 TG2 specific, yet inhibitory epitopes. Theseantibodies have been cloned, sequenced and IgG isolated with IC₅₀ valuescalculated. Three antibodies (AB1, DC1 and BB7) targeting a substratepocket proved particularly effective inhibitors. Most importantly theseantibodies also worked well both in a cell lysate and in cell cultureindicating that these antibodies have the potential to function in aprotein rich environment which is critical for in vivo application.

We believe a key element in the successful generation of theseinhibitory antibodies has been the decision to immunise with just thecore protein. To our knowledge none of the commercial TG2 antibodieshave inhibitory potential of any significance. Our own attempts to usefull length TG2 resulted in a large number of antibodies, few of whichwere specific to TG2 and none of which were inhibitory. This wouldappear to be due to a clear immunogenic preference for protein loopswithin full length TG2 many of which fall on the rear of the catalyticcore in similar positions to the most widely used anti TG2 antibody,CUB7402 (aa447 aa478).

It is surprising that our approach has led to the production of muchmore effective antibodies. Without being bound by any theory we thinkthat by simply raising antibodies to a smaller protein covering just thecentral core, we not only eliminate some of the favoured immunologicalepitopes, but we also force core targeting. This alone increases thevariety of antibodies available for selection and thus wider coverage ofthe core. However, immunising with just the core means that much of thefolding of the core is lost and thus some of the epitopes that perhapsmay be less available within a whole TG2 molecule may be more attractiveepitopes with the core in this format. Given that all 10 of theantibodies recognised linear epitopes (i.e. bound to TG2 on a reducinggel), while 80% of the antibodies we previously isolated using fulllength TG2 as an immunogen were conformation dependent, does suggestthis may be a major factor.

There have previously been other studies that have postulated the ideaof a TG2 inhibitory antibody for human application. Esposito andcolleagues developed recombinant antibodies from patients with celiacdisease where it has been postulated that TG2 antibodies may have aninhibitory role [19]. One of these antibodies was developed forcommercial application by Quark Biotechnology and a patent applicationfiled (WO2006/100679). This antibody demonstrated some exciting earlydata in the prevention of kidney fibrosis in the rat UUO model. However,we produced a recombinant version of this antibody and while it reactedwith TG2 in ELISA (FIG. 17) and western blot, we achieved littleinhibition up to 500 ng of IgG per ng of TG2 for this antibody at whichall of the antibodies developed in this study block essentially all TG2activity. Furthermore WO2006/100679 describes the generation of a mouseversion of this human antibody, and as such, long application inrecognised rat models of kidney disease would prove difficult.

Of note in the present study is the mapping of the 3 inhibitory epitopeswithin the TG2 core. The AB1 epitope is by far the most potent totarget, which is perhaps surprising given the position of the epitope.Examination of its position within the predicted TG2 active structure[20] suggests it binds in the entry port to the catalytic triad in whatmay be a substrate pocket. Given the substrates we used in our screeningassay are relatively small (putrescine and dimethyl casein), it isperhaps surprising that this site is so effective. However the positionof the epitope must be such that the large IgG (150 kDa) is positionedtightly into the catalytic site. From the epitope data one may havepredicted that the DD9 site may be more effective as it is associatedwith a putative calcium binding site [21]. However examination of theliterature suggests 5 or more putative Ca²⁺ binding sites [21] and whileit clearly has a dramatic effect, is not critical for all TG2 activity.

The DF4 site would be hypothetically the most effective epitope as theantibody binds to 1 of the essential amino acids in the catalytic triad.However it has not been possible to successfully clone out DF4 producingthis inhibitory antibody and as such the production of sufficient IgG toadequately perform IC₅₀ tests has not been possible. It may in fact bevery difficult to clone out antibodies that have too high efficacy giventhe work from Gunzler et al (1982) FEBS Lett. 150(2): 390-6 thatsuggested that lymphocytes needed TG2 activity to proliferate and thusantibodies with better inhibitory potential may only be possible usingrecombinant approaches or a continual IgG extraction system.

One of the most frustrating problems in undertaking this work has beenthe apparent inability of all antibodies developed to efficiently blocknon-human TG2 activity, which is critical for preclinical testing. Allantibodies reacted with rat and mouse TG2 in both western blot andELISA, in some cases with little difference in intensity. However out ofthe 9 antibodies we produced IgG for, it was only possible to determinean IC₅₀ for 4 in rat and none in mouse. The 4 where an IC₅₀ wascalculated against rat TG2 showed a 30 fold or lower IC₅₀ against ratTG2 than AB1 against human TG2 meaning any in vivo dose would beprohibitively large. Further none would inhibit at all in a rat celllysate. Given the reactivity in ELISA and western blots, plus there arejust 5 mismatches between species for AB1 and 3 for DD9 the significantspecies specificity for inhibition was surprising and clearlydemonstrates the critical importance of affinity for effectiveinhibition. Thus having identified these inhibitory epitopes for humanTG2 it is now critical that analogue antibodies are developed for thesesites in rat TG2 if their value is to be established in in vivo preclinical models of disease.

There are a wide range of TG inhibitors available. Notably thethiomidazole based compounds originally developed by Merke Sharpe Dome[22] the CBZ-glutamyl analogues developed by Griffin and colleagues [23]which we have used very successfully to treat experimental kidneyscarring [16] and the dihydroisoxazole type inhibitors developed byKhosla and colleagues [24-27] used successfully in various cancermodels. There has been hope that continual refinement of these compoundsmay yield a viable human TG2 inhibitor, but cross TG family reactivityor the potential toxic nature of the compounds seems to have preventedthis. More recently Acylideneoxoindoles have been described as a newreversible class of TG2 inhibitors [24], but data regarding their crossreactivity to other TG family members is lacking. At the 2010 Gordonconference on TG2 in human disease Pasternack and colleagues from Zedirapresented details of a range of compounds that use side chain Michaelacceptors as TG2 inhibitors with claims of suitability for in vivoapplication and TG2 selectivity, however a full publication on these hasnot materialised to date. At the same meeting early work from Macdonaldet al demonstrated some interesting developments in designing a TG2inhibitor for treatment of Huntington's Chorea, but again a fullpublication is still awaited. Undoubtedly a small molecule inhibitor ofTG2 would be highly desirable should it be achievable. Tissuepenetration, the ability to cross the blood brain barrier, production,cost and easy dosing are just some of the benefits. However, an antibodyinhibitor as developed here may in some way be preferable.

TG2 clearly is a multifunction enzyme and has been linked to a range ofcellular functions including nuclear stabilisation and transport [28,29], endocytosis [30, 31], GTPase signalling [32-34], Apoptosis [35,36], cell adhesion [37-39], cytoskeletal integrity [28, 29] and ECMstabilisation [9]. Clearly a small molecule inhibitor may impede on allof these functions as in general they have free access to theextracellular space and cell interior. An antibody cannot enter the celland as such the intracellular roles of TG2 would not be affected.Importantly most of the pathological roles of TG2 appear to beextracellular such as its role in tissue scarring and fibrosis, celiacdisease and cancer. Thus using an antibody would bring an additionaldegree of selectivity preventing undesired intracellular effects.Therefore an antibody would offer advantages in blocking TG2 in fibroticand scarring diseases where TG2 crosslinks ECM proteins, in celiacdisease where gliadin is deamidated in the extracellular space and inchemo-resistance in cancer where cell adhesion appears to be theprotective factor. However, unless a small Fab fragment could bedesigned that could cross the blood brain barrier a TG2 inhibitingantibody would be little use in treating neurological pathologies.

In conclusion, for the first time we have been able to develop TG2inhibitory antibodies that selectively target TG2. We have alsoidentified 3 novel inhibitory epitopes within the core domain of TG2.Humanisation of antibody AB1 will open up the possibility for the firsttime of targeted TG2 therapy in man.

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EXAMPLE 2: SEQUENCING OF NOVEL TG2 INHIBITORY ANTIBODIES OF THEINVENTION Antibody Sequencing

In order to establish the sequences of the variable regions of eachantibody of the invention, a pellet of the hybridoma cells was processedusing the Qiagen RNeasy Mini Kit to extract the RNA following themanufacturer's protocols. The extracted RNA was reverse transcribed toproduce a cDNA using a 1^(st) Strand cDNA Synthesis Kit (GE Healthcare),using a Notl-dT₁₈ primer, in accordance with the manufacturer'sprotocols. The cDNA preparation was cleaned up using the Qiagen PCRPurification Kit, in accordance with the manufacturer's protocols.

To determine the heavy chain sequence, the mouse cDNA was amplified byPCR using a set of degenerate primers (MHV1-12) with a constant regionprimer (MHCG1, MHCG2A, MHCG2B, MHCG3, or a mixture of the four) as shownin Table 12. Similarly, to determine the light chain sequence, the mousecDNA was amplified using a set of degenerate primers (MVK1-11) with aconstant region primer MKC as shown in Table 13.

If no amplification products were seen using the initial set of HeavyChain PCR a 5′ RACE PCR (Invitrogen) was carried out, using theNotl-dT₁₈ primer to generate cDNA; and the constant region primers(MHCG1, MHCG2A, MHCG2B, MHCG3, or a mixture of the four) and the 5′ RACEAnchor Primer, GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG (where I is the basefor deoxyinosine) for the PCR.

The resulting amplification bands were ligated into the pCR2.1®-TOPO®vector using the TOPO-TA Cloning® kit (Invitrogen) using themanufacturer's protocol and sent to GATC Biotech AG for sequencing.

TABLE 12 PCR Primers for Cloning Mouse Heavy Chain Variable Regions NameSequence MHV1 ATGAAATGCAGCTGGGGCATCTTCTTC MHV2ATGGGATGGAGCTRTATCATSYTCTT MHV3 ATGAAGWTGTGGTTAAACTGGGTTTTT MHV4ATGRACTTTGGGYTCAGCTTGRTTT MHV5 ATGGACTCCAGGCTCAATTTAGTTTTCCTT MHV6ATGGCTGTCYTRGSGCTRCTCTTCTGC MHV7 ATGGRATGGAGCKGGRTCTTTMTCTT MHV8ATGAGAGTGCTGATTCTTTTGTG MHV9 ATGGMTTGGGTGTGGAMCTTGCTATTCCTG MHV10ATGGGCAGACTTACATTCTCATTCCTG MHV11 ATGGATTTTGGGCTGATTTTTTTTATTG MHV12ATGATGGTGTTAAGTCTTCTGTACCTG MHCG1 CAGTGGATAGACAGATGGGGG MHCG2ACAGTGGATAGACCGATGGGGC MHCG2b CAGTGGATAGACTGATGGGGG MHCG3CAAGGGATAGACAGATGGGGC

Ambiguity codes: R=A or G; Y=C or T; M=A or C; K=G or T; S=G or C; W=Aor T.

MHV indicates primers that hybridize to the leader sequences of mouseheavy chain variable region genes, MHCG indicates primers that hybridizeto the mouse constant region genes.

TABLE 13 PCR Primers for Cloning Mouse Kappa Light Chain VariableRegions Name Size Sequence MKV1 30-mer ATGAAGTTGVVTGTTAGGCTGTTGGTGCTGMKV2 29-mer ATGGAGWCAGACACACTCCTGYTATGGGTG MKV3 30-merATGAGTGTGCTCACTCAGGTCCTGGSGTTG MKV4 33-merATGAGGRCCCCTGCTCAGWTTYTTGGMWTCTTG MKV5 30-merATGGATTTWAGGTGCAGATTWTCAGCTTC MKV6 27-mer ATGAGGTKCKKTGKTSAGSTSCTGRGGMKV7 31-mer ATGGGCWTCAAGATGGAGTCACAKWYYCWGG MKV8 31-merATGTGGGGAYCTKTTTYCMMTTTTTCAATTG MKV9 25-mer ATGGTRTCCWCASCTCAGTTCCTTGMKV10 27-mer ATGTATATATGTTTGTTGTCTATTTCT MKV11 28-merATGGAAGCCCCAGCTCAGCTTCTCTTCC CL12A ATGRAGTYWCAGACCCAGGTCTTYRT CL12BATGGAGACACATTCTCAGGTCTTTGT CL13 ATGGATTCACAGGCCCAGGTTCTTAT CL14ATGATGAGTCCTGCCCAGTTCCTGTT CL15 ATGAATTTGCCTGTTCATCTCTTGGTGCT CL16ATGGATTTTCAATTGGTCCTCATCTCCTT CL17A ATGAGGTGCCTARCTSAGTTCCTGRG CL17BATGAAGTACTCTGCTCAGTTTCTAGG CL17C ATGAGGCATTCTCTTCAATTCTTGGG MKC 20-merACTGGATGGTGGGAAGATGG

Ambiguity codes: R=A or G; Y=C or T; M=A or C; K=G or T; S=G or C; W=Aor T.

MKV indicates primers that hybridise to leader sequences of the mousekappa light chain variable region genes, MKC indicates the primer thathybridises to the mouse kappa constant region gene.

Sequence Data

Antibody AB1 was sequenced in addition to Antibodies BB7, DC1, JE12,EH6, AG9, AH3, DD9, DH2, DD6 and IA12. The sequences are provided inFIGS. 18 to 28.

EXAMPLE 3: CONSTRUCTION AND CHARACTERISATION OF CHIMERIC AND HUMANISEDNOVEL ANTI-TG2 ANTIBODIES OF THE INVENTION

To further characterise the antibodies of the invention and to enableranking and prioritisation of antibodies for humanisation, a panel ofchimeric TG2 antibodies were constructed (murine variable regions andhuman IgG1 and human kappa). The methodology used to produce thechimeric antibodies is set out below.

Methods

Human VH and VK cDNA Databases

The protein sequences of human and mouse immunoglobulins from theInternational Immunogenetics Database 2009¹ and the Kabat DatabaseRelease 5 of Sequences of Proteins of Immunological Interest (lastupdate 17 Nov. 1999)² were used to compile a database of humanimmunoglobulin sequences in a Kabat alignment. Our database contains10,606 VH and 2,910 VK sequences.

Molecular Model of AB1

As a representative of the Group 1 antibodies (i.e. antibodies that bindthe epitope spanning amino acids 304 to 326 of human TG2), a homologymodel of the mouse antibody AB1 variable regions has been calculatedusing the Modeller program³ run in automatic mode. The atomiccoordinates of 1 MQK.pdb, 3LIZ.pdb and 1MQK.pdb were the highestidentity sequence templates for the Interface, VL and VH respectively asdetermined by Blast analysis of the Accelrys antibody pdb structuresdatabase. These templates were used to generate 20 initial models, thebest of which was refined by modeling each CDR loop with its 3 best looptemplates.

hAB1 Framework Selection

The sequence analysis program, gibsSR, was used to interrogate the humanVH and VK databases with the AB1 VHc, VKc and VKc₁, the BB7 VHc and VKcand the DC1 VHc and VKc protein sequences using various selectioncriteria. FW residues within 5 Å of a CDR residue (Kabat definition) inthe homology model of mouse antibody AB1, were identified, anddesignated as the “5 Å Proximity” residues.

AF06220 was chosen as the FW on which to base the initial humanisedheavy chain versions. Table 14 shows the alignment and residue identityof AF06220 to murine antibodies. Table 15 shows the 5 Å proximityenvelope of the sequences. AF062260 has only 1 somatic mutation awayfrom its germline VH gene Z12347 (Table 16).

AY247656 was chosen as the FW on which to base the initial AB1 humanisedkappa light chain. The alignment and residue identity to murine AB1antibody kappa light chain are shown in Table 17; Table 18 shows the 5 Åproximity envelope of the sequences. The sequence shows 5 somaticmutations from its germline VK gene X93620 (Table 19).

AF193851 was chosen as the FW on which to base the other humanised kappalight chain constructs. The alignment and residue identity to the murineantibodies are shown in Table 20. Table 21 shows the 5 Å proximityenvelope of the sequences. The sequence shows no somatic mutations fromits germline VK gene J00248 (Table 22).

Generation of Expression Vectors

Construction of chimeric expression vectors entails adding a suitableleader sequence to VH and VL, preceded by a Hind III restriction siteand a Kozak sequence. The Kozak sequence ensures efficient translationof the variable region sequence. It defines the correct AUG codon fromwhich a ribosome can commence translation, and the most critical base isthe adenine at position−3, upstream of the AUG start.

For the heavy chain, the construction of the chimeric expression vectorsentails introducing a 5′ fragment of the human γ1 constant region, up toa natural ApaI restriction site, contiguous with the 3′ end of the Jregion of the variable region. The CH is encoded in the expressionvector downstream of the inserted VH sequence but lacks the V-C intron.

For the light chain, the natural splice donor site and a BamHI site isadded downstream of the V region. The splice donor sequence facilitatessplicing out the kappa V:C intron which is necessary for in-frameattachment of the VL to the constant region.

The DNA sequences of the variable regions were optimized and synthesizedby GeneArt®. The leader sequence has been selected as one that givesgood expression of antibody in cultured mammalian cells.

Heavy Chain variable region constructs were excised from the cloningvector using HindIII+ApaI digestion, purified and ligated into thesimilarly-cut and phosphatase-treated MRCT heavy chain expressionvector, and were used to transform TOP10 bacteria.

Kappa chain variable region constructs were excised using HindIII+BamHIdigestion, purified, ligated into the similarly-cut and phosphatasetreated MRCT kappa light chain expression vector, and were used totransform TOP10 bacteria.

Antibody Expression

A double insert expression vector coding for both Heavy and kappa chainswas generated and transfected into HEK293T cells. Cell culturesupernatant was purified by affinity chromatography on Protein G-agarosein accordance with the manufacturer's protocols.

Binding ELISA

HEK 293F cells were co-transfected with combinations of differenthumanised light chain vectors in association with different humanisedheavy chain vectors. Recombinant human TG2 was used to measure antibodybinding by ELISA. The results indicated that the Heavy Chain version RHA(Table 23), in combination with either Light Chain versions RKE and RKJ(Table 24) (representing the different Light Chain versions humanised)showed optimal binding (FIG. 34), and was therefore selected for furthercharacterization. A similar approach was used to identify optimal pairsof humanized BB7 heavy and light chains, and humanised DC1 heavy andlight chains.

Heavy Chain version RHA is an un-modified graft of the mouse CDR regionsof the AB1 antibody onto the Human donor sequence. However, both LightChain versions RKE and RKJ, have the same single 5 Å proximity residebackmutation, F72 (Table 24). This back mutation lies outside theVernier⁴, Canonical⁵ or Interface⁶ residues.

REFERENCES

-   1. Lefranc, M. P. IMGT, the international ImMunoGeneTics Database®.    Nucleic Acids Res. 31, 307-310 (2003).-   2. Kabat, E. A., Wu, T. T., Perry, H. M., Gottesman, K. S. &    Foeller, C. Sequences of Proteins of Immunological Interest. NIH    National Technical Information Service, (1991).-   3. Eswar, N. et al. Comparative protein structure modeling using    Modeller. Curr. Protoc. Bioinformatics. Chapter 5: Unit 5.6., Unit    (2006).-   4. Foote, J. & Winter, G. (1992). Antibody framework residues    affecting the conformation of the hypervariable loops. J Mol. Biol.    224, 487-499.-   5. Morea, V., Lesk, A. M. & Tramontano, A. (2000). Antibody    modeling: implications for engineering and design. Methods 20,    267-279.-   6. Chothia, C., Novotny, J., Bruccoleri, R. & Karplus, M. (1985).    Domain association in immunoglobulin molecules. The packing of    variable domains. J Mol. Biol. 186, 651-663.

The following table A summarises the chimeric and humanised antibodiesproduced with a cross reference to the identifiers used in the figures.

Murine Antibody Chimeric Antibody Humanised Antibody AB1 cAB001 hAB004(hAB001AE) cAB003 hAB005 (hAB001AJ) BB7 cBB001 hBB001AA hBB001BB DC1cDC001 hDC001AA hDC001BB DD6 cDD6001 DD9 cDD9001 DH2 cDH001Tables

Table 14 showing the alignment and residue identity of AF062260 to themurine antibodies. Residue identities are shown by a dot (.) character.Residue differences are shown where applicable. Gaps (−) are used tomaintain Kabat numbering, and to show residue insertion or deletionwhere applicable.

TABLE 15 5 Å Proximity Residues AB_VHc (mAB001VH)EVQLCAFTLSWVRWVARFTISRNLYCAKWG BB7_VHc (mBB7001VH)AVQLCAIIFSWVRWVARFTVSRSLYCAKWG DC1_VHc (mDC001VHEVQLCAFTLSWVRWVARFTISRNLFCARWG AF062260 ........F......S..............

Table 15 showing the antibody heavy chain framework residues that liewithin a 5 Å envelope of the CDR's. Residue identities are shown by adot (.) character. Residue differences are shown where applicable.

Table 16 showing AF062260 has 1 somatic mutation away from the germlineVH gene Z12347. Residue identities are shown by a dot (.) character.Residue differences are shown where applicable.

Table 17 showing the alignment and residue identity of AY247656 to themurine AB1 antibody. Residue identities are shown by a dot (.)character. Residue differences are shown where applicable. Gaps (−) areused to maintain Kabat numbering, and to show residue insertion ordeletion where applicable.

TABLE 18 5Å Proximity Residues AB_VKc (mAB001VK)EIVLTQTCWFTLIYGVPFSGSGSGQDFFYCFG AY247656.........YL.............T..T....

Table 18 showing the AB1 antibody kappa light chain framework residuesthat lie within a 5 Å envelope of the CDR's. Residue identities areshown by a dot (.) character. Residue differences are shown whereapplicable.

Table 19 showing AY247656 has 5 somatic mutation away from the germlineVK gene X93620. Residue identities are shown by a dot (.) character.Residue differences are shown where applicable.

Table 20 showing the alignment and residue identity of AF193851 to themurine antibodies. Residue identities are shown by a dot (.) character.Residue differences are shown where applicable. Gaps (−) are used tomaintain Kabat numbering, and to show residue insertion or deletionwhere applicable.

TABLE 21 5 Å Proximity Residues AB_VKc1 (mAB002VK)DIQMTQTCWFTLIYGVPFSGSGSGQDFFYCFG AB1_VKc2DIQKTQTCWFTLIYGVPFSGSGSGQDFFYCFG (mAB003VK) BB7_VKc (mBB001VK)AIKMTQTCWFTLIYGVPFSGSGSGQEFLYCFG DC1_VKc (mDC001VK)DITMTQTCWFILIYGVPFSGSGSGQDYAYCFG AF193851...M......S.............T..T....

Table 21 showing the antibody kappa light chain framework residues thatlie within a 5 Å envelope of the CDR's. Residue identities are shown bya dot (.) character. Residue differences are shown where applicable.

Table 22 showing AF193851 has no somatic mutation away from the germlineVK gene J00248. Residue identities are shown by a dot (.) character.Residue differences are shown where applicable.

Table 23 showing the sequence alignments of the final humanised versionsof AB1, BB7 and DC1 heavy chains. Gaps (−) are used to maintain Kabatnumbering, and to show residue insertion or deletion where applicable.

Table 24 showing the sequence alignments of the final humanised versionsof AB1, BB7 and DC1 kappa light chains. Gaps (−) are used to maintainKabat numbering, and to show residue insertion or deletion whereapplicable.

TABLE 24A Heavy chain - CDR1 AB CAASGFTFSSSAMSWVR or FTFSSSAMSWVR orSSAMS. BB7 RHB CAASGFTFSSSAMSWVR or FTFSSSAMSWVR or SSAMS. BB7 RHACAASGIIFSSSAMSWVR or IIFSSSAMSWVR or SSAMS. DC1 RHA CAASGFTFSTHAMSWVR orFTFSTHAMSWVR or THAMS. DC1 RHB CAASGFTLSTHAMSWVR or FTLSTHAMSWVR orTHAMS. Heavy chain - CDR2 AB WVSTISVGGGKTYYPDSVKGRFTISRDNSKNTL orWVSTISVGGGKTYYPDSVKGRFTISRDN or WVSTISVGGGKTYYPDSVKGRFTISR orTISVGGGKTYYPDSVKG. BB7 RHB WVSTISSGGRSTYYPDSVKGRFTISRDNSKNTL orWVSTISSGGRSTYYPDSVKGRFTISRDN or WVSTISSGGRSTYYPDSVKGRFTISR orTISSGGRSTYYPDSVKG. BB7 RHA WVATISSGGRSTYYPDSVKGRFTVSRDSSKNTL orWVATISSGGRSTYYPDSVKGRFTVSRDS or WVATISSGGRSTYYPDSVKGRFTVSR orTISSGGRSTYYPDSVKG. DC1 RHA WVSTISSGGRSTYYPDSVKGRFTISRDNSKNTL orWVSTISSGGRSTYYPDSVKGRFTISRDN or WVSTISSGGRSTYYPDSVKGRFTISR orTISSGGRSTYYPDSVKG. DC1 RHB WVATISSGGRSTYYPDSVKGRFTISRDNSKNTL orWVATISSGGRSTYYPDSVKGRFTISRDN or WVATISSGGRSTYYPDSVKGRFTISR orTISSGGRSTYYPDSVKG. Heavy chain - CDR3 AB YCAKLISLYWG or LISLY. BB7 RHBYCAKLISPYWG or LISPY. BB7 RHA YCAKLISPYWG or LISPY. DC1 RHA YCAKLISTYWGor LISTY. DC1 RHB FCARLISTYWG or LISTY. Light chain - CDR1 AB RKETCKASQDINSYLTWY or KASQDINSYLT. AB RKJ TCKASQDINSYLTWF or KASQDINSYLT.BB7 RKB TCKASQDINSYLTWF or KASQDINSYLT. BB7 RKA TCKASQDINSYLTWF orKASQDINSYLT. DC1 RKA TCKASQDINSYLTWF or KASQDINSYLT. DC1 RKBTCKASQDINSYLTWF or KASQDINSYLT. Light chain - CDR2 AB RKELLIYRTNRLFDGVPSRFSGSGSGTDFF or LLIYRTNRLFDGVP or RTNRLFD AB RKJSLIYRTNRLFDGVPSRFSGSGSGTDFF or SLIYRTNRLFDGVP or RTNRLFD BB7 RKBSLIYLTNRLMDGVPSRFSGSGSGTDFF or SLIYLTNRLMDGVP or LTNRLMD BB7 RKATLIYLTNRLMDGVPSRFSGSGSGQEFL or TLIYLTNRLMDGVP or LTNRLMD DC1 RKASLIYLVNRLVDGVPSRFSGSGSGTDFF or SLIYLVNRLVDGVP or LVNRLVD DC1 RKBILIYLVNRLVDGVPSRFSGSGSGQDYA or ILIYLVNRLVDGVP or LVNRLVD Light chain -CDR3 AB RKE YCLQYDDFPYTFG or LQYDDFPYT. AB RKJ YCLQYDDFPYTFG orLQYDDFPYT. BB7 RKB YCLQYVDFPYTFG or LQYVDFPYT. BB7 RKA YCLQYVDFPYTFG orLQYVDFPYT. DC1 RKA YCLQYDDFPYTFG or LQYDDFPYT. DC1 RKB YCLQYDDFPYTFG orLQYDDFPYT.

Table 24a summarises the sequence information presented in Tables 23 and24, in particular showing the sequence of the CDRs, and the CDRs withflanking regions, in the heavy- and light-chains of the AB, BB-7 andDC-1 antibodies.

Characterization of Chimeric and Humanised Antibodies

Chimeric and humanised Abs were assayed for binding to human andcynomolgus monkey TG2 and for enzymatic inhibition of these enzymesaccording to the methodology described below.

Methods ELISA Assay for TG2 Binding

Antibody binding to human and cynomolgus monkey TG2 was determined in anELISA assay. Clear polystyrene “Maxisorp” 96-well plates (Nunc) werecoated with 50 ng purified human or cynomogulus monkey TG2 in 50 μl 0.05M carbonate-bicarbonate buffer pH 9.6 at 4° C. overnight. Control wellswere coated with 50 μl 100 μg/ml bovine serum albumin (BSA). Plates werewashed 3× with 300 μl phosphate-buffered saline pH7.4 (PBS) containing0.1% Tween 20 (PBST) and blocked with 300 μl 3% w/v Marvel skimmed milkin PBS for 1 hour at room temperature. After 3× wash with PBST, 50 μlprotein-A purified chimeric or humanised anti-TG2 antibodies or humanIgG1 kappa isotope control antibody or CUB7402 (Abcam) were seriallydiluted 4-fold from a top concentration of 50 nM in PBS, and added tothe plate in duplicate. After 1 hour at room temperature, the plateswere washed 3× in PBST and incubated with 50 μl peroxidase-conjugatedgoat anti-human IgG (Fc) (Serotec) diluted 1/5,000 in 3% w/v Marvelskimmed milk in PBS or for wells containing CUB7402peroxidase-conjugated 1/5,000 goat anti-mouse IgG (Fc) (Sigma) for 1hour at room temperature. After 3× washes with PBST, the plates weredeveloped with 50 μl TMB substrate (Sigma) for 5 min at room temperaturebefore stopping the reaction with 25 μl 0.5M H2SO4 and readingabsorbance at 450 nM in a microtiter plate reader (BioTek EL808). Doseresponse curves were analysed and EC50 values and other statisticalparameters determined using a 4-parameter logistical fit of the data(GraphPad Prism).

Fluorescence-Based Transglutaminase Assay of TG2 Inhibition byAntibodies of the Invention.

Transglutaminase activities of purified human (Zedira) or cynomogulusmonkey TG2 enzymes (Trenzyme) were measured by incorporation ofdansylated lysine KxD (Zedira) into N,N-dimethylated casein (DMC,Sigma). Human or cynomogulus monkey TG2 were diluted in transamidationbuffer (25 mM HEPES pH 7.4 containing 250 mM NaCl, 2 mM MgCl₂, 5 mMCaCl₂, 0.2 mM DTT and 0.05% v/v Pluronic F-127) to 1 nM and 10 nMrespectively and mixed with various concentrations of protein-A purifiedmurine, chimeric or humanised TG2 antibodies for 180 min at roomtemperature in 384-well black microtiter plates (Corning). Reactionswere initiated by addition of DMC and KxD to a final concentration of 10uM and 20 uM respectively and a final reaction volume of 30 ul, andallowed to proceed at RT for 180 min and the increase in fluorescence(RFU) (excitation at 280 nm, emission 550 nm) monitored using a TecanSafire² plate reader. Data were normalised to percentage activity where% activity=(RFU test antibody−RFU low controls)/RFU high controls−RFUlow controls)×100, where low controls contained all components exceptenzyme and high controls contained all components except antibody.

Antibody dose response curves were plotted using GraphPad prism softwareand fitted using a 4-parameter logistical model to return IC50 and otherstatistical parameters. The results are illustrated in FIGS. 29 to 33.

Results and Discussion of Enzyme Inhibition and ELISA BindingExperiments by Humanized and Murine Anti-TG2 Antibodies

The ability of chimeric and humanized TG2 antibodies to inhibittransamidation by human TG2 was determined by dose-dependent inhibitionof TG2-dependent incorporation of dansylated lysine intoN,N-dimethylated casein (exemplified in FIGS. 29 and 31). Both chimericand humanized antibodies from group 1 (e.g. cAB003, cBB001, cDC001,hBB001AA, hAB001BB, hAB005 and hAB004) show potent inhibition of TG2activity in the low nanomolar range, consistent with ELISA data thatshows binding to immobilized human TG2 in the same range (FIGS. 35 and37). In contrast, the commercial antibody CUB7402 failed to inhibithuman or cynomogulus monkey TG2 enzymatic activities (FIGS. 29F and30B), despite comparable binding to group 1 antibodies in the ELISAassays (FIGS. 35A, 36A, 37A and 38A) consistent with recognition byCUB7402 of an epitope that does not interfere with the transamidationfunction of the enzyme. Therefore group 1 antibodies can bedistinguished by their ability to inhibit enzyme function from otherantibodies such as CUB7402, which bind but have no effect on enzymaticactivity. Similarly, murine and chimeric antibodies representative ofgroup 3 (e.g. mDD9001, mDH001, cDD9001 and cDH001) consistentlyinhibited human TG2 transamidation, but with lower potency than group 1antibodies (FIGS. 29 and 33). Inhibition of human TG2 by exemplifiedparent murine monoclonal antibodies from group 1 and group 3 (FIG. 33)show comparable potencies to their chimeric and humanized versions,indicating that the functional potency of the murine antibodies has beenretained in the humanized versions. Similarly the human TG2 ELISAbinding data for the exemplified humanized antibodies hBB001AA, hBB001BBand hAB004 (FIG. 37) show comparable EC50 values with those obtained forthe chimeric versions cBB001 and cAB003, indicating that bindingaffinity has also been preserved in the humanized versions. Chimeric andhumanized antibodies also demonstrate potent inhibition of cynomogulusmonkey TG2 (FIGS. 30 and 32) and comparable ELISA EC50's (FIGS. 36 and38) across the species, consistent with the conservation of the cognateepitope in cynomogulus monkey TG2, which has overall 99% sequenceidentity. In contrast CUB7402 shows comparable binding to TG2 of bothspecies as the group 1 antibodies, but inhibits neither enzyme activity(FIGS. 35-38 and 29-30).

Cell Based Assays

Binding of antibodies of the invention to extracellular TG2 from HK-2epithelial cells was assayed using the following protocol.

Measurement of Extracellular TG Activity

Extracellular TG activity was measured by modified cell ELISA. HK-2epithelial cells were harvested using Accutase and plated at a densityof 2×10⁴ cells/well in serum free medium onto a 96 well plate that hadbeen coated overnight with 100 μl/well of fibronectin (5 μg/ml in 50 mMTris-HCl pH 7.4) (Sigma, Poole UK). Cells were allowed to attach for O/Nat 37° C. Media was replaced with DMEM (Life Technologies) andcompounds, antibodies or controls were added and allowed to bind at 37°C. 0.1 mM biotin cadaverine [N-(5 amino pentyl biotinamide)trifluoroacetic acid] (Zedira) was added to wells and the plate returnedto 37° C. for 2 hours. Plates were washed twice with 3 mM EDTA/PBS andcells removed with 0.1% (w/v) deoxycholate in 5 mM EDTA/PBS. Thesupernatant was collected and used for protein determination. Plateswere washed with PBS/Tween and incorporated biotin cadaverine revealedusing 1:5000 extravidin HRP (Sigma, Poole, UK) for 1 h at roomtemperature followed by K Blue substrate (SkyBio). The reaction wasstopped with Red Stop (SkyBio) and the absorbance read at 4 650 nm. Eachantibody was tested on at least three separate occasions.

Results are provided in FIGS. 39 and 40 and show an exemplar curve andtable of IC50 values obtained for the antibodies tested. FIG. 39displays the results with Humanised AB1 and FIG. 40 displays the resultswith Humanised BB7.

hAB005 inhibited the extracellular TG2 of the HK2 cells with an IC50 of71.85 nM and a maximal inhibition of about 30% control activity.hBB001AA inhibited the activity with an IC50 of 19.8 nM and a maximalinhibition of 40% control activity. hBB001BB had a better IC50 of 4.9 nMbut a maximal inhibition of about 55% control.

Scratch Assays

Scratch wound assays were also performed to assess binding activity ofhumanised and or chimeric anti-TG2 antibodies of the invention.

TG2 has been shown to have an important role in lung fibrosis and TG2knockout mice show reduced scarring and fibrosis in the bleomycin model.(Keith C. Olsen, Ramil E. Sapinoro, R. M. Kottmann, Ajit A. Kulkarni,Siiri E. Iismaa, Gail V. W. Johnson, Thomas H. Thatcher, Richard P.Phipps, and Patricia J. Sime. (2011) Transglutaminase 2 and Its Role inPulmonary Fibrosis. Am. J of Respiratory & Critical Care Med. 1840699-707) Migration of cells from TG2 knockout mice on wounding wasreduced compared to wild type. Scratch wound assay were performed toassess the effect of humanised and/or chimeric anti-TG2 antibodies ofthe invention on the rate of wound closure in a layer of normal lungfibroblasts (WI-38 cells).

Scratch Assay Protocol:

WI-38 cells (normal human lung fibroblasts ATCC cat#CCL-75) were platedin a 96 well Image Lock plate (Essen cat#4379) at 2×10⁴/well in αMEMmedia (Life Technologies cat#32561) with 10% FBS and grown O/N to >97%confluence. Cells were washed 2× with αMEM media without serum and ascratch wound was generated using an Essen Wound Maker and themanufacturers protocol. The media was removed and replaced with 95μl/well serum free media. Controls and test antibodies were added to thewells. The plate was placed in an Essen Incucyte and the closure of thewound was analysed using the scratch wound protocol.

Cytochalasin D was used as an assay control at 0.1 μM. R281, a smallmolecule non-specific transglutaminase inhibitor, was tested at 100 μM.Z DON, a peptide non reversible transglutaminase inhibitor was tested at10 μM and 100 μM. The commercially available TG2 antibody Cub7402 (ABcamcat#ab2386) was tested at 5 μg/ml. Antibodies of the invention weretested on at least three occasions at various concentrations asindicated. In all experiments controls were Cytochalasin D at 0.1 uM andZDON at two concentrations to show a dose dependant effect.

Exemplar results of the scratch assays are shown in FIGS. 41 to 44. Ascan be seen in FIG. 41, Cytochalasin D, R281 and ZDON all inhibitedwound closure (ZDON was shown to inhibit in a dose dependant manner) butthe antibody Cub7402 did not inhibit wound closure. Humanised BB7,Humanised AB1 and Chimeric DC1 all inhibited wound closure.

Affinities of Chimeric and Humanised Anti TG2 Abs

The binding affinities (Kds and off rates) for a panel of chimeric andhumanised Abs of the invention against human TG2 and cyano TG2 wereassessed using Biacore techniques. The protocols and results aredescribed below and shown in FIGS. 45 to 47.

Biacore Methods

Recombinant human TG2 was obtained from Zedira GmbH (cat. no.: T002).Recombinant cynomolgus monkey TG2 was obtained from Trenzyme. Surfaceplasmon resonance (SPR) was measured on a Biacore T200 instrument (GEHealthcare). CM5 chips (GE Healthcare cat. no.: BR-1006-68) were coatedwith monoclonal mouse anti-human IgG1 (Fc) (MAH) antibody (GE Healthcarecat. no.: BR-1008-39) by amine-coupling as described in themanufacturers instructions. HBS-EP+ buffer (0.01 M HEPES, 0.15 M NaCl, 3mM EDTA, 0.05% v/v Surfactant P20) and HBS-P+ buffer (0.01 M HEPES, 0.15M NaCl, 0.05% Surfactant P20) were purchased from GE Healthcare as 10×stocks (cat. nos.: BR-1006-69 and BR-1006-71). Calcium Chloride solutionwas obtained from Sigma Aldrich (cat. no.: 21115).

The method employed to determine the affinity of the anti-TG2 antibodiesinvolved the capture of the chimeric or humanised antibodies on a MAHcoated CM5 chip, followed by the injection of a series of TG2 samples inrunning buffer. The running buffer was 1× HBS-P+ containing 1 mM CaCl₂,or 1×HBS-EP+ for calcium-free experiments. Antibody capture was carriedout for a contact time of 120 seconds at a flow rate of 10 μl/minresulting in the capture of approximately 40-80 RU. TG2 was injectedover the immobilised antibody at concentrations ranging from 25 nM to400 nM with a contact time up to 600 seconds at a flow rate of 30μl/min. Dissociation of TG2 was typically measured for up to 5400seconds (1.5 hours). Regeneration of the chip was then performed using 3M MgCl₂, for a contact time of 60 seconds at a flow rate of 30 μl/ml,followed by a 300s stabilisation period before the next sample. For eachof human and cynomolgus monkey TG2, at least 5 injections at a varietyof concentrations were performed in at least two separate experiments.

Kinetic data were exported from the Biacore T200 Evaluation Software andanalysed using GraphPad Prism, where the association phases anddissociation phases were analysed separately using a one phaseassociation model and one-phase exponential decay model respectively.Association rates (k_(on)) were calculated for each curve individually,and dissociation rates (k_(off)) values from the long dissociation phasedata collected. Where k_(off) values were calculated to be <1×10⁻⁵ s⁻¹,values were set at 1×10⁻⁵ for analysis, as rates slower than this couldnot be estimated accurately. Values for k_(on) and k_(off) are presentedin the tables below as the mean of the individual calculated values foreach antibody for each TG2 species from multiple concentrations +/−1standard deviation. K_(D) values are calculated as mean k_(off)/meank_(on).

Results of Biacore Experiments

TABLE 25 Human TG2 Antibody k_(off) (s⁻¹) st. dev k_(on) (M⁻¹s⁻¹) st.dev K_(D) (M) cAB003 +Ca²⁺ <10⁻⁵ — 1.7 × 10⁵ 3.2 × 10⁴  <6 × 10⁻¹¹ −Ca²⁺<10⁻⁵ — 8.6 × 10⁴ 2.1 × 10⁴  <1 × 10⁻¹⁰ cBB001 +Ca²⁺ <10⁻⁵ — 2.1 × 10⁵6.9 × 10⁴  <5 × 10⁻¹¹ −Ca²⁺ <10⁻⁵ — 1.5 × 10⁵ 1.8 × 10⁴  <7 × 10⁻¹¹hAB004 (hAB001AE) 2.4 × 10⁻⁵ 1.0 × 10⁻⁵ 2.0 × 10⁵ 1.4 × 10⁵ 1.2 × 10⁻¹⁰hAB005 (hAB001AJ) <10⁻⁵ — 1.9 × 10⁵ 6.8 × 10⁴  <5 × 10⁻¹¹ hBB001AA <10⁻⁵— 2.7 × 10⁵ 1.1 × 10⁵  <4 × 10⁻¹¹ hBB001BB <10⁻⁵ — 2.4 × 10⁵ 1.2 × 10⁵ <4 × 10⁻¹¹ cDC001 <10⁻⁵ — 3.2 × 10⁵ 5.7 × 10⁴  <3 × 10⁻¹¹ cDH001 +Ca²⁺1.8 × 10⁻⁵ 4.9 × 10⁻⁶ 2.8 × 10⁴ 1.3 × 10⁴ 6.4 × 10⁻¹⁰ −Ca²⁺ 4.9 × 10⁻⁴1.6 × 10⁻⁵ 2.1 × 10⁴ 1.2 × 10⁴ 2.3 × 10⁻⁸  cDD9001 +Ca²⁺ 1.3 × 10⁻⁵ 1.8× 10⁻⁶ 3.0 × 10⁴ 2.3 × 10⁴ 4.3 × 10⁻¹⁰ −Ca²⁺ 7.1 × 10⁻⁵ 1.6 × 10⁻⁵ 2.3 ×10⁴ 7.6 × 10³ 3.1 × 10⁻⁹ 

Table 25 shows the kinetic data obtained against human TG2. Wherek_(off) rates were calculated to be less than 10⁻⁵ s⁻¹, values were setto 10⁵ s⁻¹ for analysis, as rates slower than this could not beaccurately determined.

TABLE 26 Cynomolgus TG2 Antibody k_(off) (s⁻¹) st. dev k_(on) (M⁻¹s⁻¹)st. dev K_(D) (M) cAB003 +Ca²⁺ 1.2 × 10⁻⁵ 8.1 × 10⁻⁶ 2.4 × 10⁵ 1.1 × 10⁵5.0 × 10⁻¹¹ −Ca²⁺ 1.3 × 10⁻⁵ 1.5 × 10⁻⁶ 1.4 × 10⁵ 1.9 × 10⁴ 9.3 × 10⁻¹¹cBB001 +Ca²⁺ <10⁻⁵ — 2.9 × 10⁵ 9.0 × 10⁴  <3 × 10⁻¹¹ −Ca²⁺ <10⁻⁵ — 1.9 ×10⁵ 2.1 × 10⁴  <5 × 10⁻¹¹ hAB004 (hAB001AE) 1.8 × 10⁻⁵ 2.3 × 10⁻⁶ 1.6 ×10⁵ 7.3 × 10⁴ 1.1 × 10⁻¹¹ hAB005 (hAB001AJ) 3.4 × 10⁻⁵ 4.6 × 10⁻⁶ 1.4 ×10⁵ 4.1 × 10⁴ 2.4 × 10⁻¹⁰ hBB001AA <10⁻⁵ — 3.0 × 10⁵ 1.4 × 10⁵  <3 ×10⁻¹¹ hBB001BB 3.7 × 10⁻⁵ 3.4 × 10⁻⁶ 2.7 × 10⁵ 1.3 × 10⁵ 1.4 × 10⁻¹⁰cDC001 <10⁻⁵ — 4.2 × 10⁵ 3.3 × 10⁵ 2.4 × 10⁻¹¹ cDH001 +Ca²⁺ <10⁻⁵ — 1.5× 10⁴ 9.5 × 10³  <7 × 10⁻¹⁰ −Ca²⁺ 6.3 × 10⁻⁵ 1.2 × 10⁻⁵ 2.6 × 10⁴ 1.0 ×10⁴ 2.4 × 10⁻⁹  cDD9001 +Ca²⁺ 1.6 × 10⁻⁵ 1.4 × 10⁻⁵ 3.1 × 10⁴ 1.6 × 10⁴5.2 × 10⁻¹⁰ −Ca²⁺ 4.1 × 10⁻⁵ 1.6 × 10⁻⁵ 4.6 × 10⁴ 1.3 × 10⁴ 8.9 × 10⁻¹⁰

Table 26 shows the kinetic data obtained against cynomolgus TG2. Wherek_(off) rates were calculated to be less than 10⁻⁵ s⁻¹, values were setto 10⁻⁵ s⁻¹ for analysis, as rates slower than this could not beaccurately determined.

FIGS. 45 to 47 provide example Biacore data sets. As can be seen, thehumanised and chimeric antibodies cAB003, cBB001, hAB004, hAB005,hBB001AA, hBB001BB, cDC001 for TG2 have excellent affinity for human andcynomolgus TG2, with K_(D) values of 120 pM or better. The chimericantibodies cDH001 and cDD9001 exhibit slower association rates to humanand cynomolgus TG2 and weaker overall affinity. Examination of aselection of antibodies in the absence of calcium shows that there islittle or no effect, except in the case of cDH001 and cDD9001, wherebinding is weaker due to faster dissociation rates (k_(off)).

1. A method of inhibiting human transglutaminase type 2 (TG2)-mediatedcross-linking of lysine and glutamine with Nε(γ-glutamyl)lysineisopeptide bonds, the method comprising contacting human TG2 with anantibody that selectively binds to an epitope within amino acids 143-473of human TG2.
 2. (canceled)
 3. The method of claim 1, wherein theantibody selectively binds to a region comprising: i) amino acids 304 to326 of human TG2; or ii) amino acids 351 to 365 of human TG2; or iii)amino acids 450 to 467 of human TG2.
 4. (canceled)
 5. The method ofclaim 1, wherein the antibody comprises, or one or more amino acidsequence having at least 90% identity with one or more of the followingamino acid sequences: (SEQ ID NO: 7) KASQDINSYLT or (SEQ ID NO: 24)TCKASQDINSYLTWF or (SEQ ID NO: 37) TCKASQDINSYLTWY; and/or (SEQ ID NO:8) RTNRLFD or (SEQ ID NO: 25) TLIYRTNRLFDGVP or (SEQ ID NO: 26)TLIYRTNRLFDGVPXXFSGSGSGQDFF or (SEQ ID NO: 38) LLIYRTNRLFDGVP or (SEQ IDNO: 39) SLIYRTNRLFDGVP or (SEQ ID NO: 40) LLIYRTNRLFDGVPXXFSGSGSGQDFF or(SEQ ID NO: 41) SLIYRTNRLFDGVPXXFSGSGSGQDFF or (SEQ ID NO: 13) LTNRLMDor (SEQ ID NO: 17) LVNRLVD or (SEQ ID NO: 76) XXNRLXD or (SEQ ID NO:229) LLIYRTNRLFDGVPSRFSGSGSGTDFF or (SEQ ID NO: 230)SLIYRTNRLFDGVPSRFSGSGSGTDFF or (SEQ ID NO: 231)SLIYLTNRLMDGVPSRFSGSGSGTDFF or (SEQ ID NO: 233)TLIYLTNRLMDGVPSRFSGSGSGQEFL or (SEQ ID NO: 235)SLIYLVNRLVDGVPSRFSGSGSGTDFF or (SEQ ID NO: 237)ILIYLVNRLVDGVPSRFSGSGSGQDYA or (SEQ ID NO: 234) TLIYLTNRLMDGVP or (SEQID NO: 232) SLIYLTNRLMDGVP or (SEQ ID NO: 236) SLIYLVNRLVDGVP or (SEQ IDNO: 17) LVNRLVD or (SEQ ID NO: 238) ILIYLVNRLVDGVP; and/or (SEQ ID NO:9) LQYDDFPYT or (SEQ ID NO: 27) YCLQYDDFPYTFG or (SEQ ID NO: 14)LQYVDFPYT or (SEQ ID NO: 14) LQYVDFPYT or (SEQ ID NO: 77) LQYXDFPYT or(SEQ ID NO: 239) YCLQYVDFPYTFG; and/or (SEQ ID NO: 10) SSAMS or (SEQ IDNO: 28) FTLSSSAMSWVR or (SEQ ID NO: 29) CXAXXFTLSSSAMSWVR or (SEQ ID NO:42) FTFSSSAMSWVR or (SEQ ID NO: 43) CXAXXFTFSSSAMSWVR or (SEQ ID NO: 18)THAMS or (SEQ ID NO: 78) XXAMS or (SEQ ID NO: 207) CAASGFTFSSSAMSWVR or(SEQ ID NO: 208) CAASGIIFSSSAMSWVR or (SEQ ID NO: 209) IIFSSSAMSWVR or(SEQ ID NO: 210) CAASGFTFSTHAMSWVR or (SEQ ID NO: 211) FTFSTHAMSWVR or(SEQ ID NO: 213) CAASGFTLSTHAMSWVR or (SEQ ID NI: 214) FTLSTHAMSWVR;and/or (SEQ ID NO: 11) TISVGGGKTYYPDSVKG or (SEQ ID NI: 30)WVATISVGGGKTYYPDSVKGRFTISR or (SEQ ID NO: 31)WVATISVGGGKTYYPDSVKGRFTISRXNXXXXL or (SEQ ID NO: 44)WVSTISVGGGKTYYPDSVKGRFTISR or (SEQ ID NO: 45)WVSTISVGGGKTYYPDSVKGRFTISRXNXXXXL or (SEQ ID NO: 15) TISSGGRSTYYPDSVKGor (SEQ ID NO: 15) TISSGGRSTYYPDSVKG or (SEQ ID NO: 79)TISXGGXXTYYPDSVKG or (SEQ ID NO: 215) WVSTISVGGGKTYYPDSVKGRFTISRDNSKNTLor (SEQ ID NO: 216) WVSTISVGGGKTYYPDSVKGRFTISRDN or (SEQ ID NO: 217)WVSTISSGGRSTYYPDSVKGRFTISRDNSKNTL or (SEQ ID NO: 218)WVSTISSGGRSTYYPDSVKGRFTISRDN or (SEQ ID NO: 219)WVSTISSGGRSTYYPDSVKGRFTISR or (SEQ ID NO: 220)WVATISSGGRSTYYPDSVKGRFTVSRDSSKNTL or (SEQ ID NO: 221)WVATISSGGRSTYYPDSVKGRFTVSRDS or (SEQ ID NO: 222)WVATISSGGRSTYYPDSVKGRFTVSR or (SEQ ID NO: 223)WVATISSGGRSTYYPDSVKGRFTISRDNSKNTL or (SEQ ID NO: 224)WVATISSGGRSTYYPDSVKGRFTISRDN or (SEQ ID NO: 225)WVATISSGGRSTYYPDSVKGRFTISR; and/or (SEQ ID NO: 12) VLISLY or (SEQ ID NO:32) YCAKLISLYWG or (SEQ ID NO: 16) LISPY or (SEQ ID NO: 19) LISTY or(SEQ ID NO: 80) LISXY or (SEQ ID NO: 226) YCAKLISPYWG or (SEQ ID NO:227) YCAKLISTYWG or (SEQ ID NO: 228) FCARLISTYWG,

wherein X is any amino acid.
 6. (canceled)
 7. The method of claim 1,wherein the antibody has at least one light chain variable regioncomprising the amino acid sequence (SEQ ID NO: 47)DIKMTQSPSSMYASLGERVTITCKASQDINSYLTWFQQKPGKSPKTLIYRTNRLFDGVPSRFSGSGSGQDFFLTISSLEYEDMGIYYCLQYDDFPYTFGG GTKLEIK, or (SEQ IDNO: 46) DIQMTQTPSSMYASLGERVTITCKASQDINSYLTWFQQKPGKSPKTLIYRTNRLFDGVPSRFSGSGSGQDFFLTISSLEYEDMGIYYCLQYDDFPYTFGG GTKLEIK, or (SEQ IDNO: 48) EIVLTQSPSSMYASLGERVTITCKASQDINSYLTWFQQKPGKSPKTLIYRTNRLFDGVPSRFSGSGSGQDFFLTISSLEYEDMGIYYCLQYDDFPYTFGG GTKLEIK (AB-1_VK), or(SEQ ID NO: 49) DIQMTQSPSSMYASLGERVTITCKASQDINSYLTWFQQKPGKSPKTLIYRTNRLFDGVPSRFSGSGSGQDFFLTISSLEYEDMGIYYCLQYDDFPYTFGG GTKLEIK (AB-1_VK1),or (SEQ ID NO: 50) EIVLTQSPSSLSASVGDRVTITCKASQDINSYLTWYQQKPGKAPKLLIYRTNRLFDGVPSRFSGSGSGTDFFFTISSLQPEDFGTYYCLQYDDFPYTFGG GTKLEIK (hAB-1_RKE),or (SEQ ID NO: 51) DIQMTQSPSSLSASVGDRVTITCKASQDINSYLTWFQQKPGKAPKSLIYRTNRLFDGVPSRFSGSGSGTDFFLTISSLQPEDFATYYCLQYDDFPYTFGQ GTKVEIK (hAB-1_RKJ),or (SEQ ID NO: 60) AIKMTQSPSSMYASLGERVIITCKASQDINSYLTWFQQKPGKSPKTLIYLTNRLMDGVPSRFSGSGSGQEFLLTISGLEHEDMGIYYCLQYVDFPYTFGG GTKLEIK (BB-7 VK), or(SEQ ID NO: 70) DITMTQSPSSIYASLGERVTITCKASQDINSYLTWFQQKPGKSPKILIYLVNRLVDGVPSRFSGSGSGQDYALTISSLEYEDMGIYYCLQYDDFPYTFGG GTKLEIK (DC-1 VK), or(SEQ ID NO: 87) QIVLTQSPAIMSASPGEKVTMTCSASSSVDYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMGAEDAATYYCQQWNSSPLTFGAG TKLELK (DD-9 VK), or(SEQ ID NO: 89) QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATFYCQQWSSSPLTFGAG TKLELK (DH-2 VK), or(SEQ ID NO: 62) DIQMTQSPSSLSASVGDRVTITCKASQDINSYLTWFQQKPGKAPKSLIYLTNRLMDGVPSRFSGSGSGTDFFLTISSLQPEDFATYYCLQYVDFPYTFGQ GTKVEIK, or (SEQ IDNO: 63) DIKMTQSPSSLSASVGDRVTITCKASQDINSYLTWFQQKPGKAPKTLIYLTNRLMDGVPSRFSGSGSGQEFLLTISSLQPEDFATYYCLQYVDFPYTFGQ GTKVEIK, or (SEQ IDNO: 72) DIQMTQSPSSLSASVGDRVTITCKASQDINSYLTWFQQKPGKAPKSLIYLVNRLVDGVPSRFSGSGSGTDFFLTISSLQPEDFATYYCLQYDDFPYTFGQ GTKVEIK, or (SEQ IDNO: 73) DITMTQSPSSLSASVGDRVTITCKASQDINSYLTWFQQKPGKAPKILIYLVNRLVDGVPSRFSGSGSGQDYALTISSLQPEDFATYYCLQYDDFPYTFGQ GTKVEIK; and/or atleast one heavy chain variable region comprising the amino acid sequence(SEQ ID NO: 53) EVQLVESGGGLVKPGGSLKLSCAASGFTLSSSAMSWVRQTPDRRLEWVATISVGGGKTYYPDSVKGRFTISRDNAKNTLYLQMNSLRSEDTAMYYCAKLI SLYWGQGTTLTVSS, or(SEQ ID NO: 52) EVQLEESGGGLVKPGGSLKLSCAASGFTLSSSAMSWVRQTPDRRLEWVATISVGGGKTYYPDSVKGRFTISRDNAKNTLYLQMNSLRSEDTAMYYCAKLI SLYWGQGTTLTVSS, or(SEQ ID NO: 54) EVQLQESGGGLVKPGGSLKLSCAASGFTLSSSAMSWVRQTPDRRLEWVATISVGGGKTYYPDSVKGRFTISRDNAKNTLYLQMNSLRSEDTAMYYCAKLI SLYWGQGTTLTVSS(AB-1_VH), or (SEQ ID NO: 55)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSSAMSWVRQAPGKGLEWVSTISVGGGKTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKLI SLYWGQGTLVTVSS(hAB-1_RHA), or (SEQ ID NO: 61)AVQLVESGGGLVKPGGSLKLSCAASGIIFSSSAMSWVRQTPEKRLEWVATISSGGRSTYYPDSVKGRFTVSRDSAKNTLYLQMDSLRSEDTAIYYCAKLI SPYWGQGTTLTVSS (BB-7VH), or (SEQ ID NO: 71)EVQLVESGGGLVKPGGSLKLSCAASGFTLSTHAMSWVRQTPEKRLEWVATISSGGRSTYYPDSVKGRFTISRDNVKNTLYLQLSSLRSEDTAVYFCARLI STYWGQGTTLTVSS (DC-1VH), or (SEQ ID NO: 88)QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVSWIRQSSGKGLEWLAHIYWDDDKRYNPSLKSRITISKDSSSNQVFLKITSVDTADTATYYCARS WTTAPFAFWGQGTLVTVSA(DD-9VH), or (SEQ ID NO: 90)QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWLAHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITSVDTADTATYYCARS GTTAPFAYWGQGTLVTVSA(DH-2VH), or (SEQ ID NO: 64)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSSAMSWVRQAPGKGLEWVSTISSGGRSTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKLI SPYWGQGTLVTVSS, or(SEQ ID NO: 65) EVQLLESGGGLVQPGGSLRLSCAASGIIFSSSAMSWVRQAPGKGLEWVATISSGGRSTYYPDSVKGRFTVSRDSSKNTLYLQMNSLRAEDTAVYYCAKLI SPYWGQGTLVTVSS, or(SEQ ID NO: 74) EVQLLESGGGLVQPGGSLRLSCAASGFTFSTHAMSWVRQAPGKGLEWVSTISSGGRSTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKLI STYWGQGTLVTVSS, or(SEQ ID NO: 75) EVQLLESGGGLVQPGGSLRLSCAASGFTLSTHAMSWVRQAPGKGLEWVATISSGGRSTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCARLI STYWGQGTLVTVSS,

or an amino acid sequence having at least 90% identity with one or moreof the amino acid sequences specified above.
 8. The method of claim 1,wherein the antibody comprises one or more of the following amino acidsequences: (SEQ ID NO: 7) KASQDINSYLT or (SEQ ID NO: 24) TCKASQDINSYLTWFor (SEQ ID NO: 37) TCKASQDINSYLTWY; and/or (SEQ ID NO: 8) RTNRLFD or(SEQ ID NO: 25) TLIYRTNRLFDGVP or (SEQ ID NO: 26)TLIYRTNRLFDGVPXXFSGSGSGQDFF or (SEQ ID NO: 38) LLIYRTNRLFDGVP or (SEQ IDNO: 39) SLIYRTNRLFDGVP or (SEQ ID NO: 40) LLIYRTNRLFDGVPXXFSGSGSGQDFF or(SEQ ID NO: 41) SLIYRTNRLFDGVPXXFSGSGSGQDFF or (SEQ ID NO: 13) LTNRLMDor (SEQ ID NO: 17) LVNRLVD or (SEQ ID NO: 76) XXNRLXD or (SEQ ID NO:229) LLIYRTNRLFDGVPSRFSGSGSGTDFF or (SEQ ID NO: 230)SLIYRTNRLFDGVPSRFSGSGSGTDFF or (SEQ ID NO: 231)SLIYLTNRLMDGVPSRFSGSGSGTDFF or (SEQ ID NO: 233)TLIYLTNRLMDGVPSRFSGSGSGQEFL or (SEQ ID NO: 235)SLIYLVNRLVDGVPSRFSGSGSGTDFF or (SEQ ID NO: 237)ILIYLVNRLVDGVPSRFSGSGSGQDYA or (SEQ ID NO: 234) TLIYLTNRLMDGVP or (SEQID NO: 232) SLIYLTNRLMDGVP or (SEQ ID NO: 236) SLIYLVNRLVDGVP or (SEQ IDNO: 17) LVNRLVD or (SEQ ID NO: 238) ILIYLVNRLVDGVP; and/or (SEQ ID NO:9) LQYDDFPYT or (SEQ ID NO: 27) YCLQYDDFPYTFG or (SEQ ID NO: 14)LQYVDFPYT or (SEQ ID NO: 14) LQYVDFPYT or (SEQ ID NO: 77) LQYXDFPYT or(SEQ ID NO: 239) YCLQYVDFPYTFG; and/or (SEQ ID NO: 10) SSAMS or (SEQ IDNO: 28) FTLSSSAMSWVR or (SEQ ID NO: 29) CXAXXFTLSSSAMSWVR or (SEQ ID NO:42) FTFSSSAMSWVR or (SEQ ID NO: 43) CXAXXFTFSSSAMSWVR or (SEQ ID NO: 18)THAMS or (SEQ ID NO: 78) XXAMS or (SEQ ID NO: 207) CAASGFTFSSSAMSWVR or(SEQ ID NO: 208) CAASGIIFSSSAMSWVR or (SEQ ID NO: 209) IIFSSSAMSWVR or(SEQ ID No: 210) CAASGFTFSTHAMSWVR or (SEQ ID NO: 211) FTFSTHAMSWVR or(SEQ ID NO: 213) CAASGFTLSTHAMSWVR or (SEQ ID NI: 214) FTLSTHAMSWVR;and/or (SEQ ID NO: 11) TISVGGGKTYYPDSVKG or (SEQ ID NO: 30)WVATISVGGGKTYYPDSVKGRFTISR or (SEQ ID NO: 31)WVATISVGGGKTYYPDSVKGRFTISRXNXXXXL or (SEQ ID NO: 44)WVSTISVGGGKTYYPDSVKGRFTISR or (SEQ ID NO: 45)WVSTISVGGGKTYYPDSVKGRFTISRXNXXXXL or (SEQ ID NO: 15) TISSGGRSTYYPDSVKGor (SEQ ID NO: 15) TISSGGRSTYYPDSVKG or (SEQ ID NO: 79)TISXGGXXTYYPDSVKG or (SEQ ID NO: 215) WVSTISVGGGKTYYPDSVKGRFTISRDNSKNTLor (SEQ ID NO: 216) WVSTISVGGGKTYYPDSVKGRFTISRDN or (SEQ ID NO: 217)WVSTISSGGRSTYYPDSVKGRFTISRDNSKNTL or (SEQ ID No: 218)WVSTISSGGRSTYYPDSVKGRFTISRDN or (SEQ ID NO: 219)WVSTISSGGRSTYYPDSVKGRFTISR or (SEQ ID NO: 220)WVATISSGGRSTYYPDSVKGRFTVSRDSSKNTL or (SEQ ID NO: 221)WVATISSGGRSTYYPDSVKGRFTVSRDS or (SEQ ID NO: 222)WVATISSGGRSTYYPDSVKGRFTVSR or (SEQ ID NO: 223)WVATISSGGRSTYYPDSVKGRFTISRDNSKNTL or (SEQ ID NO: 224)WVATISSGGRSTYYPDSVKGRFTISRDN or (SEQ ID NO: 225)WVATISSGGRSTYYPDSVKGRFTISR; and/or (SEQ ID NO: 12) LISLY or (SEQ ID NO:32) YCAKLISLYWG or (SEQ ID NO: 16) LISPY or (SEQ ID NO: 19) LISTY or(SEQ ID NO: 80) LISXY or (SEQ ID NO: 226) YCAKLISPYWG or (SEQ ID NO:227) YCAKLISTYWG or (SEQ ID NO: 228) FCARLISTYWG,

wherein X is any amino acid.
 9. The method of claim 8, wherein theantibody has at least one light chain variable region comprising anamino acid sequence having at least 90% identity with the followingamino acid sequences: (SEQ ID NO: 81) (K/Q)ASQDI(N/S)(S/N)YL(T/N), (SEQID NO: 7) for example KASQDINSYLT; and (SEQ ID NO: 82)(R/L/V/D)(T/V/A)(N/S)(R/N)L(F/M/V/E)(D/T), (SEQ ID NO: 8) for exampleRTNRLFD; and (SEQ ID NO: 83) (L/Q)QY(D/V/N)(D/T)(F/Y)P(Y/L)T, (SEQ IDNO: 9) for example LQYDDFPYT;

and/or at least one heavy chain variable region comprising an amino acidsequence having at least 90% identity with the following amino acidsequences: (SEQ ID NO: 84) (S/T)(S/H/Y)AMS, (SEQ ID NO: 10) for exampleSSAMS; and (SEQ ID NO: 85) (T/A)IS(V/S/G)(G/S)G(G/R)(K/S)TYY(P/A)DSVKG,(SEQ ID NO: 11) for example TISVGGGKTYYPDSVKG; and (SEQ ID NO: 86)(L/D)(I/G)(S/G)(L/P/T/V)Y, (SEQ ID NO: 12) for example LISLY.


10. The method of claim 9, wherein the antibody has at least one lightchain variable region comprising the amino acid sequence (SEQ ID NO: 47)DIKMTQSPSSMYASLGERVTITCKASQDINSYLTWFQQKPGKSPKTLIYRTNRLFDGVPSRFSGSGSGQDFFLTISSLEYEDMGIYYCLQYDDFPYTFGG GTKLEIK, or (SEQ IDNO: 46) DIQMTQTPSSMYASLGERVTITCKASQDINSYLTWFQQKPGKSPKTLIYRTNRLFDGVPSRFSGSGSGQDFFLTISSLEYEDMGIYYCLQYDDFPYTFGG GTKLEIK, or (SEQ IDNO: 48) EIVLTQSPSSMYASLGERVTITCKASQDINSYLTWFQQKPGKSPKTLIYRTNRLFDGVPSRFSGSGSGQDFFLTISSLEYEDMGIYYCLQYDDFPYTFGG GTKLEIK (AB-1_VK), or(SEQ ID NO: 49) DIQMTQSPSSMYASLGERVTITCKASQDINSYLTWFQQKPGKSPKTLIYRTNRLFDGVPSRFSGSGSGQDFFLTISSLEYEDMGIYYCLQYDDFPYTFGG GTKLEIK (AB-1_VK1),or (SEQ ID NO: 50) EIVLTQSPSSLSASVGDRVTITCKASQDINSYLTWYQQKPGKAPKLLIYRTNRLFDGVPSRFSGSGSGTDFFFTISSLQPEDFGTYYCLQYDDFPYTFGG GTKLEIK (hAB-1_RKE),or (SEQ ID NO: 51) DIQMTQSPSSLSASVGDRVTITCKASQDINSYLTWFQQKPGKAPKSLIYRTNRLFDGVPSRFSGSGSGTDFFLTISSLQPEDFATYYCLQYDDFPYTFGQ GTKVEIK (hAB-1_RKJ),or (SEQ ID NO: 60) AIKMTQSPSSMYASLGERVIITCKASQDINSYLTWFQQKPGKSPKTLIYLTNRLMDGVPSRFSGSGSGQEFLLTISGLEHEDMGIYYCLQYVDFPYTFGG GTKLEIK (BB-7 VK), or(SEQ ID NO: 70) DITMTQSPSSIYASLGERVTITCKASQDINSYLTWFQQKPGKSPKILIYLVNRLVDGVPSRFSGSGSGQDYALTISSLEYEDMGIYYCLQYDDFPYTFGG GTKLEIK (DC-1 VK), or(SEQ ID NO: 87) QIVLTQSPAIMSASPGEKVTMTCSASSSVDYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMGAEDAATYYCQQWNSSPLTFGAG TKLELK (DD-9 VK), or(SEQ ID NO: 89) QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATFYCQQWSSSPLTFGAG TKLELK (DH-2 VK), or(SEQ ID NO: 62) DIQMTQSPSSLSASVGDRVTITCKASQDINSYLTWFQQKPGKAPKSLIYLTNRLMDGVPSRFSGSGSGTDFFLTISSLQPEDFATYYCLQYVDFPYTFGQ GTKVEIK, or (SEQ IDNO: 63) DIKMTQSPSSLSASVGDRVTITCKASQDINSYLTWFQQKPGKAPKTLIYLTNRLMDGVPSRFSGSGSGQEFLLTISSLQPEDFATYYCLQYVDFPYTFGQ GTKVEIK, or (SEQ IDNO: 72) DIQMTQSPSSLSASVGDRVTITCKASQDINSYLTWFQQKPGKAPKSLIYLVNRLVDGVPSRFSGSGSGTDFFLTISSLQPEDFATYYCLQYDDFPYTFGQ GTKVEIK, or (SEQ IDNO: 73) DITMTQSPSSLSASVGDRVTITCKASQDINSYLTWFQQKPGKAPKILIYLVNRLVDGVPSRFSGSGSGQDYALTISSLQPEDFATYYCLQYDDFPYTFGQ GTKVEIK; and/or atleast one heavy chain variable region comprising the amino acid sequence(SEQ ID NO: 53) EVQLVESGGGLVKPGGSLKLSCAASGFTLSSSAMSWVRQTPDRRLEWVATISVGGGKTYYPDSVKGRFTISRDNAKNTLYLQMNSLRSEDTAMYYCAKLI SLYWGQGTTLTVSS, or(SEQ ID NO: 52) EVQLEESGGGLVKPGGSLKLSCAASGFTLSSSAMSWVRQTPDRRLEWVATISVGGGKTYYPDSVKGRFTISRDNAKNTLYLQMNSLRSEDTAMYYCAKLI SLYWGQGTTLTVSS, or(SEQ ID NO: 54) EVQLQESGGGLVKPGGSLKLSCAASGFTLSSSAMSWVRQTPDRRLEWVATISVGGGKTYYPDSVKGRFTISRDNAKNTLYLQMNSLRSEDTAMYYCAKLI SLYWGQGTTLTVSS(AB-1_VH), or (SEQ ID NO: 55)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSSAMSWVRQAPGKGLEWVSTISVGGGKTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKLI SLYWGQGTLVTVSS(hAB-1_RHA), or (SEQ ID NO: 61)AVQLVESGGGLVKPGGSLKLSCAASGIIFSSSAMSWVRQTPEKRLEWVATISSGGRSTYYPDSVKGRFTVSRDSAKNTLYLQMDSLRSEDTAIYYCAKLI SPYWGQGTTLTVSS(BB-7VH), or (SEQ ID NO: 71)EVQLVESGGGLVKPGGSLKLSCAASGFTLSTHAMSWVRQTPEKRLEWVATISSGGRSTYYPDSVKGRFTISRDNVKNTLYLQLSSLRSEDTAVYFCARLI STYWGQGTTLTVSS(DC-1VH), or (SEQ ID NO: 88)QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVSWIRQSSGKGLEWLAHIYWDDDKRYNPSLKSRITISKDSSSNQVFLKITSVDTADTATYYCARS WTTAPFAFWGQGTLVTVSA(DD-9VH), or (SEQ ID NO: 90)QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWLAHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITSVDTADTATYYCARS GTTAPFAYWGQGTLVTVSA(DH-2VH), or (SEQ ID NO: 64)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSSAMSWVRQAPGKGLEWVSTISSGGRSTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKLI SPYWGQGTLVTVSS, or(SEQ ID NO: 65) EVQLLESGGGLVQPGGSLRLSCAASGIIFSSSAMSWVRQAPGKGLEWVATISSGGRSTYYPDSVKGRFTVSRDSSKNTLYLQMNSLRAEDTAVYYCAKLI SPYWGQGTLVTVSS, or(SEQ ID NO: 74) EVQLLESGGGLVQPGGSLRLSCAASGFTFSTHAMSWVRQAPGKGLEWVSTISSGGRSTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKLI STYWGQGTLVTVSS, or(SEQ ID NO: 75) EVQLLESGGGLVQPGGSLRLSCAASGFTLSTHAMSWVRQAPGKGLEWVATISSGGRSTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCARLI STYWGQGTLVTVSS,

or an amino acid sequence having at least 90% identity with one or moreof the amino acid sequences specified above.
 11. The method of claim 1,wherein the antibody comprises or consists of an intact antibody. 12.The method of claim 1, wherein the antibody is an antigen-bindingfragment selected from the group consisting of: an Fv fragment, forexample a single chain Fv fragment or a disulphide-bonded Fv fragment;an Fab fragment; and an Fab-like fragment, for example an Fab′ fragmentor an F(ab)₂ fragment. 13-14. (canceled)
 15. The method according toclaim 1, wherein the antibody thereof does not inhibit TG1, TG3, TG13and/or TG7 activity. 16-24. (canceled)
 25. The method of claim 43wherein the individual has a disorder selected from the group consistingof Celiac disease, abnormal wound healing, scarring, and hypertrophicscars, ocular scarring, inflammatory bowel disease, maculardegeneration, psoriasis, fibrosis-related diseases (e.g. liver fibrosis,pulmonary fibrosis such as interstitial lung disease and fibrotic lungdisease, cardiac fibrosis, myelofibrosis, kidney fibrosis such asglomerulosclerosis and tubulointerstitial fibrosis), atherosclerosis,restenosis, inflammatory diseases, autoimmune diseases,neurodegenerative/neurological diseases (e.g. Huntington's Disease,Alzheimer's disease, Parkinson's disease, polyglutamine disease,spinobulbar muscular atrophy, dentatorubral-pallidoluysian atrophy,spinocerebellar ataxias 1, 2, 3, 6, 7 and 12, rubropallidal atrophy,spinocerebellar palsy), glioblastomas such as glioblastoma inLi-Fraumeni syndrome and sporadic glioblastoma, malignant melanomas,pancreatic ductal adenocarcinomas, myeloid leukemia, acute myelogenousleukemia, myelodysplastic syndrome, myeloproliferative syndrome,gynaecological cancer, Kaposi's sarcoma, Hansen's disease, andcollagenous colitis). 26-34. (canceled)
 35. The method of claim 1,wherein the antibody has at least one light chain variable regioncomprising an amino acid sequence having at least 90% identity with thefollowing amino acid sequences: (SEQ ID NO: 297)(K/Q/R)ASQ(D/G)I(N/S/R)(S/N)YL(T/N/A), for example (SEQ ID NO: 7)KASQDINSYLT; and (SEQ ID NO: 298)(R/L/V/D/A)(T/V/A)(N/S)(R/N)L(F/M/V/E/Q)(D/T/S), for example (SEQ ID NO:8) RTNRLFD; and (SEQ ID NO: 299) (L/Q)Q(Y/H)(D/V/N)(D/T)(F/Y)P(Y/L/W)T,for example (SEQ ID NO: 9) LQYDDFPYT;

and/or at least one heavy chain variable region comprising an amino acidsequence having at least 90% identity with the following amino acidsequences: (SEQ ID NO: 300) (S/T)(S/H/Y)AMS, for example (SEQ ID NO: 10)SSAMS; and (SEQ ID NO: 301) (T/A)IS(V/S/G)(G/S)G(G/R)(K/S)TYY(P/A)DSVKG,for example (SEQ ID NO: 11) TISVGGGKTYYPDSVKG; and (SEQ ID NO: 302)(L/D)(I/G)(S/G)(L/P/T/V)Y, for example (SEQ ID NO: 12) LISLY.


36. The method of claim 8, wherein the antibody has: a light chainvariable region comprising the sequence (SEQ ID NO: 73)DITMTQSPSSLSASVGDRVTITCKASQDINSYLTWFQQKPGKAPKILIYLVNRLVDGVPSRFSGSGSGQDYALTISSLQPEDFATYYCLQYDDFPYTFGQ GTKVEIK, and a heavychain variable region comprising the sequence (SEQ ID NO: 75)EVQLLESGGGLVQPGGSLRLSCAASGFTLSTHAMSWVRQAPGKGLEWVATISSGGRSTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCARLI STYWGQGTLVTVSS,

or amino acid sequences having at least 90% identity therewith; or alight chain variable region comprising the sequence (SEQ ID NO: 63)DIKMTQSPSSLSASVGDRVTITCKASQDINSYLTWFQQKPGKAPKTLIYLTNRLMDGVPSRFSGSGSGQEFLLTISSLQPEDFATYYCLQYVDFPYTFGQ GTKVEIK, and a heavychain variable region comprising the sequence (SEQ ID NO: 65)EVQLLESGGGLVQPGGSLRLSCAASGIIFSSSAMSWVRQAPGKGLEWVATISSGGRSTYYPDSVKGRFTVSRDSSKNTLYLQMNSLRAEDTAVYYCAKLI SPYWGQGTLVTVSS,

or amino acid sequences having at least 90% identity therewith; or alight chain variable region comprising the sequence (SEQ ID NO: 50)EIVLTQSPSSLSASVGDRVTITCKASQDINSYLTWYQQKPGKAPKLLIYRTNRLFDGVPSRFSGSGSGTDFFFTISSLQPEDFGTYYCLQYDDFPYTFGG GTKLEIK, or (SEQ IDNO: 51) DIQMTQSPSSLSASVGDRVTITCKASQDINSYLTWFQQKPGKAPKSLIYRTNRLFDGVPSRFSGSGSGTDFFLTISSLQPEDFATYYCLQYDDFPYTFGQ GTKVEIK, and a heavychain variable region comprising the sequence (SEQ ID NO: 55)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSSAMSWVRQAPGKGLEWVSTISVGGGKTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKLI SLYWGQGTLVTVSS,

or amino acid sequences having at least 90% identity therewith.
 37. Themethod of claim 8, wherein the antibody comprises the following aminoacid sequences: (LCDR1; SEQ ID NO: 7) KASQDINSYLT; (LCDR2; SEQ ID NO:17) LVNRLVD; (LCDR3; SEQ ID NO: 9) LQYDDFPYT; (HCDR1; SEQ ID NO: 18)THAMS; (HCDR2; SEQ ID NO: 15) TISSGGRSTYYPDSVKG; and (HCDR3; SEQ ID NO:19) LISTY; or (LCDR1; SEQ ID NO: 7) KASQDINSYLT; (LCDR2; SEQ ID NO: 13)LTNRLMD; (LCDR3; SEQ ID NO: 14) LQYVDFPYT; (HCDR1; SEQ ID NO: 10) SSAMS;(HCDR2; SEQ ID NO: 15) TISSGGRSTYYPDSVKG; and (HCDR3; SEQ ID NO: 16)LISPY; or (LCDR1; SEQ ID NO: 7) KASQDINSYLT; (LCDR2; SEQ ID NO: 8)RTNRLFD; (LCDR3; SEQ ID NO: 9) LQYDDFPYT; (HCDR1; SEQ ID NO: 10) SSAMS;(HCDR2; SEQ ID NO: 11) TISVGGGKTYYPDSVKG; and (HCDR3; SEQ ID NO: 12)LISLY.


38. The method of claim 1, wherein the antibody has: a light chainvariable region comprising the sequence (SEQ ID NO: 73)DITMTQSPSSLSASVGDRVTITCKASQDINSYLTWFQQKPGKAPKILIYLVNRLVDGVPSRFSGSGSGQDYALTISSLQPEDFATYYCLQYDDFPYTFGQ GTKVEIK, and a heavychain variable region comprising the sequence (SEQ ID NO: 75)EVQLLESGGGLVQPGGSLRLSCAASGFTLSTHAMSWVRQAPGKGLEWVATISSGGRSTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCARLI STYWGQGTLVTVSS, or alight chain variable region comprising the sequence (SEQ ID NO: 63)DIKMTQSPSSLSASVGDRVTITCKASQDINSYLTWFQQKPGKAPKTLIYLTNRLMDGVPSRFSGSGSGQEFLLTISSLQPEDFATYYCLQYVDFPYTFGQ GTKVEIK, and a heavychain variable region comprising the sequence (SEQ ID NO: 65)EVQLLESGGGLVQPGGSLRLSCAASGIIFSSSAMSWVRQAPGKGLEWVATISSGGRSTYYPDSVKGRFTVSRDSSKNTLYLQMNSLRAEDTAVYYCAKLI SPYWGQGTLVTVSS, or alight chain variable region comprising the sequence (SEQ ID NO: 50)EIVLTQSPSSLSASVGDRVTITCKASQDINSYLTWYQQKPGKAPKLLIYRTNRLFDGVPSRFSGSGSGTDFFFTISSLQPEDFGTYYCLQYDDFPYTFGG GTKLEIK, or (SEQ IDNO: 51) DIQMTQSPSSLSASVGDRVTITCKASQDINSYLTWFQQKPGKAPKSLIYRTNRLFDGVPSRFSGSGSGTDFFLTISSLQPEDFATYYCLQYDDFPYTFGQ GTKVEIK, and a heavychain variable region comprising the sequence (SEQ ID NO: 55)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSSAMSWVRQAPGKGLEWVSTISVGGGKTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKLI SLYWGQGTLVTVSS.


39. The method of claim 1, wherein the antibody is an IgG1, IgG2, IgG3or IgG4.
 40. The method of claim 1, wherein the antibody orantigen-binding fragment has: (a) a light chain variable regioncomprising the sequenceDVLMTQNPLSLPVSLGDQASISCRSSQSIEHINGNTYLEWYLQKPGQSPKFLIYKVSNRFSGVPDRFSGSGSGTDFTLRISRVEAEDLGIYYCFQGSHVP FTFGGGTKLEIK, and aheavy chain variable region comprising the sequenceEVQLQQSGPELVKPGASVKMSCKASGYRFTSYVMHWVKQKSGQGLEWIGY INPYNDGAKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARL SSDYWGQGTTLTVSS; or (b) a lightchain variable region comprising the sequenceDVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVGAEDLGVYYCLQVSHVP FTFGSGTKLEIK, and aheavy chain variable region comprising the sequenceEVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGFINPYNDGTKYNEKFKGKATLTSDKASTTAYMELSSLTSEDSAVYYCARFS SGYWGQGTTLTVSS; or(c) a light chain variable region comprising the sequenceDVLMTQNPLSLPVSLGDQASISCRSSRSIEHSNGNTYLEWYLQKPGQSPKFLIYKVSNRFSGVPDRFSGSGSGTDFTLRISSVEAEDLGVYYCFQGSHVP FTFGGGTKLEIK, and aheavy chain variable region comprising the sequenceEVQLQQSGPELVKPGASVKMSCRASGYTFTTYVIHWVKQKPGQGLEWIGYINPYNDGARYNEKFKGKATLTSDKSSTTAYMELSSLTSEDSAVYYCARLS SDYWGQGTTLTVSS; or(d) a light chain variable region comprising the sequenceQIVLTQSPAIMSASPGEKVTMTCSASSSVDYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMGAEDAATYYCQQWNSSPLTFGAG TKLELK, and a heavychain variable region comprising the sequenceQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVSWIRQSSGKGLEWLAHIYWDDDKRYNPSLKSRITISKDSSSNQVFLKITSVDTADTATYYCARS WTTAPFAFWGQGTLVTVSA;or (e) a light chain variable region comprising the sequenceQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATFYCQQWSSSPLTFGAG TKLELK, and a heavychain variable region comprising the sequenceQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWLAHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITSVDTADTATYYCARS GTTAPFAYWGQGTLVTVSA;or (f) a light chain variable region comprising the sequenceQAVVTQESALTTSPGETVTLTCRSSTGAVAANNYANWIQEKPDHLFTGLIAGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNYWVF GGGTKVTVLG, and aheavy chain variable region comprising the sequenceEVQLQQSGPELVKPGASVKMSCKASGYRFTDYNMHWVKQNLGKSLEWIGYINPKNGVIYYNQKFKGKATLTVNRSSNTAYMEIRSLTSEDSAVYYCATAL TYWGQGTLVTVSA; or (g)a light chain variable region comprising the sequenceDVVMTQTPLTLSVTFGQPASISCKSSQSLLYDNGKTYLHWLFQRPGQSPRRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFP YTFGGGTKLEIK, and aheavy chain variable region comprising the sequenceQIQLVQSGPELKKPGETVKISCKASGYTFTTYGMTWVKQAPGKGLKWMGWINTSSGVPTYADDFKGRFAFSLETSASTAYLQINNLKSEDTATYFCARPE VAYWGQGTLVTVSA.


41. The method of claim 1, that is an in vivo method.
 42. A method oftreating fibrosis in an individual comprising administering an antibodyor antigen-binding fragment thereof that selectively binds to an epitopewithin amino acids 143-473 of human TG2.
 43. A method of inhibitinghuman transglutaminase type 2 (TG2)-mediated cross-linking of lysine andglutamine with Nε(γ-glutamyl)lysine isopeptide bonds in an individual,the method comprising administering to an individual in need thereof anantibody that selectively binds to an epitope within amino acids 143-473of human TG2.